Systems and methods for obtaining samples using ingestible devices

ABSTRACT

Ingestible devices configured to obtain samples when present in the GI tract of a subject, as well as related systems and methods, are disclosed. Sampling systems that include an absorbent material a preservative, such as an analyte preservative, as well as related materials and methods, are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119 to U.S.Provisional Application No. 62/376,688 filed on Aug. 18, 2016 and U.S.Provisional Application No. 62/545,129 filed on Aug. 14, 2017.

INCORPORATION BY REFERENCE

This application incorporates by reference the following patentapplications: U.S. Ser. Nos. 14/460,893; 15/514,413; 62/376,688;62/385,344; 62/385,553; 62/478,955; 62/434,188; 62/434,320; 62/431,297;62/434,797; 62/480,187; 62/502,383; 62/540,873; and 62/545,129.

FIELD

The disclosure relates to ingestible devices configured to obtainsamples when present in the GI tract of a subject, as well as relatedsystems and methods. The disclosure also relates to sampling systemsthat include an absorbent material a preservative, such as an analytepreservative.

BACKGROUND

The gastrointestinal (GI) tract generally contains a wealth ofinformation regarding an individual's body. For example, contents in theGI tract may provide information regarding the individual's metabolism.An analysis of the contents of the GI tract may also provide informationfor identifying relationships between the GI content composition (e.g.,relationship between bacterial and biochemical contents) and certaindiseases and disorders.

SUMMARY

In one general aspect, the disclosure provides ingestible devices thatcan obtain a sample when in the GI tract of a subject. The devices aredesigned to provide a high degree of control over when and where asample is taken. The devices can be designed to allow analysis/assayingof the sample while the device is still present in the subject, and/orcan be designed for the sample to be analyzed/assayed after the deviceexits the subject. The devices allow for careful control over the amountof sample that is taken in by the device. The disclosure also providesrelated systems and methods.

In another general aspect, the disclosure relates to sampling systemsthat include an absorbent material and a preservative, such as ananalyte preservative. The sampling systems can be configured to fitwithin an ingestible device. For example, a sampling system can be anintegral portion of an ingestible device. The disclosure also providesrelated systems and methods.

In one general aspect, the disclosure provides an ingestible devicehaving an opening between an interior of the ingestible device and anexterior of the ingestible device. The ingestible device includes achamber, and a multi-stage valve system in the interior of theingestible device. The multi-stage valve system has first, second andthird states. The first state of the multi-stage valve system isdifferent from the second and third states of the multi-stage valvesystem. The second state of the multi-stage valve system is differentfrom the first and third states of the multi-stage valve system. Whenthe multi-stage valve system is in its first state, the opening preventsfluid communication between the interior of the ingestible device andthe exterior of the ingestible device. When the multi-stage valve systemis in its second state, the opening allows fluid communication betweenthe interior of the ingestible device and the exterior of the ingestibledevice. When the multi-stage valve system is in its third state, theopening prevents fluid communication between the interior of theingestible device and the exterior of the ingestible device.

In some embodiments, the multi-stage valve system includes an actuatorsystem having first, second and third states. When the actuator systemis in its first state, the multi-stage valve system is in its firststate. When the actuator system is in its second state, the multi-stagevalve system is in its second state. When the actuator system is in itsthird state, the multi-stage valve system is in its third state.

In some embodiments, the actuator system includes first and secondmembers.

In some embodiments, when the multi-stage valve system is in its firststage, the first member holds the multi-stage system in its first state,and, when the multi-stage system is in its second stage, the secondmember holds the valve multi-stage system in its second state.

In some embodiments, the first member includes a first chamber includingwax, and the second member includes a second chamber including wax.

In some embodiments, when the actuator system is in its first state, thewax in the first chamber is solid, and, when the actuator system isconfigured so that, when the actuator is changing from its first stateto its second stage in its second state, at least a portion of the waxin the first chamber is liquid.

In some embodiments, the ingestible further includes a device configuredto heat the wax in the first chamber.

In some embodiments, when the actuator system is in its second state,the wax in the second chamber is solid, and, when the actuator system isconfigured so that, when the actuator is changing from its second stateto its second stage in its third state, at least a portion of the wax inthe second chamber is liquid.

In some embodiments, the ingestible device further includes a deviceconfigured to heat the wax in the second chamber.

In some embodiments, the multi-stage valve system further includes atrigger mechanically coupled with the actuator system.

In some embodiments, the trigger has first, second and third states.When the actuator system is in its first state, the trigger is in itsfirst state. When the actuator system is in its second state, thetrigger is in its second state. When the actuator system is in its thirdstate, the trigger is in its third state.

In some embodiments, the valve system further includes a gatemechanically coupled to the actuator system.

In some embodiments, the gate has first, second and third states. Whenthe actuator system is in its first state, the gate is in its firststate. When the actuator system is in its second state, the gate is inits second state. When the actuator system is in its third state, thegate is in its third state.

In some embodiments, the gate has an opening. When the gate is in itsfirst state, the opening of gate and the opening of the ingestibledevice are not aligned. When the gate is in its second state, theopening of gate and the opening of the ingestible device are aligned.When the gate is in its third state, the opening of gate and the openingof the ingestible device are not aligned.

In some embodiments, the multi-stage valve system further includes abiasing system mechanically coupled to actuator system.

In some embodiments, the biasing system includes first and secondbiasing members.

In some embodiments, the first member includes a first spring, and thesecond member includes a second spring.

In some embodiments, the ingestible device further includes a samplingsystem configured so that, when the valve system is in its second stage,the exterior of the ingestible device is in fluid communication with thesampling system.

In some embodiments, the sampling system includes a plurality ofabsorbent members.

In some embodiments, the sampling system includes a biomarkerpreservative.

In some embodiments, the ingestible device further includes ananalytical system configured to analyze a sample in the interior of theingestible device.

In some embodiments, the ingestible device further includes amicroprocessor configured to control at least one system of theingestible device.

In one general aspect, the disclosure provides an ingestible devicehaving an opening between an interior of the ingestible device and anexterior of the ingestible device. The ingestible device includes achamber, and a multi-stage valve system in the interior of theingestible device. The multi-stage valve system includes: an actuatorsystem including a first member; a trigger including a first peg and afirst lip; a gate including a protrusion, and a gate leg having anopening; and a biasing system including first and second biasingmembers. When the multi-stage valve system is in a first stage: thefirst biasing member applies a force to the trigger so that the firstpeg contacts the first member; the first member opposes the forceapplied to the trigger by the first biasing member; the second biasingmember applies a force to the gate so that the protrusion contacts thefirst lip; the first lip opposes the force applied to the gate by thesecond biasing member; and the opening in the gate leg is not alignedwith the opening in the ingestible device.

In some embodiments, when the multi-stage valve system is in a secondstage different from the first stage: the first lip does not contact theprotrusion; and the opening in the gate leg is aligned with the openingin the ingestible device.

In some embodiments, a position of the trigger is different when themulti-stage valve system is in its second stage compared to when themulti-stage valve system is in its first stage.

In some embodiments, a position of the gate is different when themulti-stage valve system is in its second stage compared to when themulti-stage valve system is in its first stage.

In some embodiments the actuator system includes a second member, andthe trigger includes a second peg and a second lip. When the multi-stagevalve system is in a second stage different from the first stage: thefirst biasing member applies a force to the trigger so that the secondpeg contacts the second member; the second member opposes the forceapplied to the trigger by the first biasing member; the second biasingmember applies a force to the gate so that the protrusion contacts thesecond lip; the second lip opposes the force applied to the gate by thesecond biasing member; and the opening in the gate leg is aligned withthe opening in the ingestible device.

In some embodiments, when the multi-stage valve system is in a thirdstage different from the first and second stages: the second lip doesnot contact the protrusion; and the opening in the gate leg is notaligned with the opening in the ingestible device.

In some embodiments, a position of the trigger is different when themulti-stage valve system is in its third stage compared to when themulti-stage valve system is in its first stage and its second stage.

In some embodiments, a position of the gate is different when themulti-stage valve system is in its third stage compared to when themulti-stage valve system is in its second stage.

In some embodiments, the ingestible device further includes a samplingsystem configured so that, when the valve system is in its second stage,the exterior of the ingestible device is in fluid communication with thesampling system.

In some embodiments, the sampling system includes a plurality ofabsorbent members.

In some embodiments, the sampling system includes a biomarkerpreservative.

In some embodiments, the ingestible device further includes ananalytical system configured to analyze a sample in the interior of theingestible device.

In some embodiments, the ingestible device further includes amicroprocessor configured to control at least one system of theingestible device.

In one general aspect, the disclosure provides an ingestible devicehaving an opening between an interior of the ingestible device and anexterior of the ingestible device. The ingestible device includes achamber, and a sampling system in the interior of the ingestible device.The sampling system includes a first absorbent member, and a secondabsorbent member different from the first absorbent member. The samplingsystem is configured so that fluid that flows from the exterior of theingestible device to the interior of the ingestible device enters thefirst absorbent member. The sampling system is configured to allow fluidto flow from the first absorbent member to the second absorbent member.

In some embodiments, the second absorbent member has a first end and asecond end opposite the first end. The sampling system is configured toallow fluid to flow from the first absorbent member to the first end ofthe second absorbent member.

In some embodiments, the ingestible device further includes a thirdabsorbent member different from the first and second absorbent members.

In some embodiments, the sampling system is configured to allow fluid toflow from the second absorbent member to the third absorbent member.

In some embodiments, the sampling system is configured to: prevent fluidfrom flowing directly from the first end of the second member to thethird member; and allow fluid to flow from second end of the secondabsorbent member to the third absorbent member.

In some embodiments, the ingestible device further includes a blockingmember between the second and third absorbent members, wherein theblocking member is configured to prevent the flow of fluid from thesecond absorbent to the third absorbent member.

In some embodiments, the sampling system further includes a fourthabsorbent member different from the first, second and third absorbentmembers, and the sampling system is configured to allow fluid to flowfrom the second absorbent member to the fourth absorbent member.

In some embodiments, the sampling system further includes a fourthabsorbent member different from the first, second and third absorbentmembers.

In some embodiments, the sampling system includes an analytepreservative.

In some embodiments, the sampling system further includes a cell filterbetween the first and second absorbent members.

In some embodiments, further includes a multi-stage valve system in theinterior of the ingestible device.

In some embodiments, the multi-stage valve system has first, second andthird states. The first state of the multi-stage valve system isdifferent from the second and third states of the multi-stage valvesystem. The second state of the multi-stage valve system is differentfrom the first and third states of the multi-stage valve system. Whenthe multi-stage valve system is in its first state, the opening preventsfluid communication between the interior of the ingestible device andthe exterior of the ingestible device. When the multi-stage valve systemis in its second state, the opening allows fluid communication betweenthe interior of the ingestible device and the exterior of the ingestibledevice. When the multi-stage valve system is in its third state, theopening prevents fluid communication between the interior of theingestible device and the exterior of the ingestible device.

In some embodiments, the multi-stage valve system includes: an actuatorsystem including a first member; a trigger including a first peg and afirst lip; a gate including a protrusion, and a gate leg having anopening; and a biasing system including first and second biasingmembers. When the multi-stage valve system is in a first stage: thefirst biasing member applies a force to the trigger so that the firstpeg contacts the first member; the first member opposes the forceapplied to the trigger by the first biasing member; the second biasingmember applies a force to the gate so that the protrusion contacts thefirst lip; the first lip opposes the force applied to the gate by thesecond biasing member; and the opening in the gate leg is not alignedwith the opening in the ingestible device.

In some embodiments, the ingestible device further includes ananalytical system configured to analyze a sample in the sampling system.

In some embodiments, the ingestible device further includes amicroprocessor configured to control at least one system of theingestible device.

In one general aspect, the ingestible device has an opening between aninterior of the ingestible device and an exterior of the ingestibledevice. The ingestible device includes: a chamber; and a sampling systemin the interior of the ingestible device configured to absorb a fluidthat enters the interior of the ingestible device via the opening. Thesampling system includes an absorbent member and at least onepreservative at least partially absorbed in the absorbent member.

In some embodiments, the preservative is at least one analytepreservative.

In some embodiments, the analyte preservative includes a preservativefor at least one of a nucleic acid, a small molecule, or a protein.

In some embodiments, the analyte preservative includes a preservativefor at least one nucleic acid, small molecule, or protein that is abiomarker of at least one GI disorder.

In some embodiments, the analyte preservative is a surfactant.

In some embodiments, the analyte preservative is a stabilizer.

In some embodiments, the analyte preservative includes a member selectedfrom the group consisting of a nuclease inhibitor, an RNase inhibitor,and a protease inhibitor.

In some embodiments, the analyte preservative includes an acid having apKa of from three to seven.

In some embodiments, the analyte preservative includes a paraben.

In some embodiments, the surfactant includes polysorbate.

In some embodiments, the stabilizer includes trehalose or dextran.

In some embodiments, the paraben includes a member selected from thegroup consisting of parahydroxybenzoate, an ester of parahydroxybenzoicacid, and propyl paraben.

In some embodiments, the analyte preservative includes a proteaseinhibitor.

In some embodiments, the protease inhibitor includes a member selectedfrom the group consisting of serine protease inhibitors, metalloproteaseinhibitors, aminopeptidase inhibitors, cysteine peptidase inhibitor, andaspartyl protease inhibitors.

In some embodiments, the analyte preservative includes an acid.

In some embodiments, the analyte preservative includes at least onemember selected from the group consisting of sorbic acid and citricacid.

In some embodiments, the preservative includes at least one bacteriapreservative.

In some embodiments, the bacteria preservative reduces bacterial growthand multiplication.

In some embodiments, the bacteria preservative includes a bactericidalor bacteriostatic preservative.

In some embodiments, the bacteria preservative includes a preservativefor at least one bacterium associated with at least one GI disorder.

In some embodiments, the bacteria preservative includes a memberselected from the group consisting of sorbic acid, citric acid, propylparaben, nisin, dimethyl dicarbonate, ethylenediaminetetraacetic acid(EDTA), sodium azide, hydroxyurea, fusidic acid, diazolidinyl urea,imidazolidinyl urea, salicylic acid, barium and nickle chloride,metallic copper, thimerosal, 2-phenoxyethanol, and ProClin.

In some embodiments, the bacteria preservative is sorbic acid,thimerosal, 2-phenoxyethanol, diazolinidyl urea, or imidazolinidyl urea.

In some embodiments, the absorbent member includes at least one analytepreservative in addition to the at least one bacteria preservative.

In some embodiments, the analyte preservative is a nucleic acidpreservative.

In some embodiments, the nucleic acid preservative is a DNAse inhibitoror an RNase inhibitor.

In some embodiments, the sampling system includes a plurality ofdifferent preservatives.

In some embodiments, the sampling system includes: a first absorbentmember; and a second absorbent member different from the first absorbentmember. The sampling system is configured so that fluid that flows fromthe exterior of the ingestible device to the interior of the ingestibledevice enters the first absorbent member. The sampling system isconfigured to allow fluid to flow from the first absorbent member to thesecond absorbent member.

In some embodiments, the sampling system further includes a cell filterbetween the first and second absorbent members.

In some embodiments, the sampling system further includes a cell filter.

In some embodiments, the ingestible further includes an analyticalsystem configured to analyze a sample in the sampling system.

In some embodiments, the ingestible further includes a microprocessorconfigured to control at least one system of the ingestible device.

In some embodiments, the ingestible further includes a multi-stage valvesystem in the interior of the ingestible device.

In some embodiments: the multi-stage valve system has first, second andthird states; the first state of the multi-stage valve system isdifferent from the second and third states of the multi-stage valvesystem; the second state of the multi-stage valve system is differentfrom the first and third states of the multi-stage valve system; whenthe multi-stage valve system is in its first state, the opening preventsfluid communication between the interior of the ingestible device andthe exterior of the ingestible device; when the multi-stage valve systemis in its second state, the opening allows fluid communication betweenthe interior of the ingestible device and the exterior of the ingestibledevice; and when the multi-stage valve system is in its third state, theopening prevents fluid communication between the interior of theingestible device and the exterior of the ingestible device.

In some embodiments, the multi-stage valve system includes: an actuatorsystem including a first member; a trigger including a first peg and afirst lip; a gate including a protrusion, and a gate leg having anopening; and a biasing system including first and second biasingmembers. When the multi-stage valve system is in a first stage: thefirst biasing member applies a force to the trigger so that the firstpeg contacts the first member; the first member opposes the forceapplied to the trigger by the first biasing member; the second biasingmember applies a force to the gate so that the protrusion contacts thefirst lip; the first lip opposes the force applied to the gate by thesecond biasing member; and the opening in the gate leg is not alignedwith the opening in the ingestible device.

In some embodiments, the ingestible device further includes ananalytical system configured to analyze a sample in the sampling system.

In one general aspect, the disclosure provides a method, that includescollecting a sample into a sampling system of an ingestible device. Thesampling system includes an absorbent member and at least onepreservative at least partially absorbed in the absorbent member.

In some embodiments, the ingestible device is an ingestible device is aningestible device as disclosed herein.

In one general aspect, the disclosure provides a sampling system thatincludes: an absorbent member; and at least one preservative at leastpartially absorbed in the absorbent member. The absorbent member isconfigured to absorb a fluid.

In some embodiments, the preservative is at least one analytepreservative.

In some embodiments, the analyte preservative includes a preservativefor at least one of a nucleic acid, a small molecule, or a protein.

In some embodiments, the analyte preservative includes a preservativefor at least one nucleic acid, small molecule, or protein that is abiomarker of at least one GI disorder.

In some embodiments, the analyte preservative is a surfactant.

In some embodiments, the analyte preservative is a stabilizer.

In some embodiments, the analyte preservative includes a member selectedfrom the group consisting of a nuclease inhibitor, an RNase inhibitor,and a protease inhibitor.

In some embodiments, the analyte preservative includes an acid having apKa of from three to seven.

In some embodiments, the analyte preservative includes a paraben.

In some embodiments, the surfactant includes polysorbate.

In some embodiments, the stabilizer includes trehalose or dextran.

In some embodiments, the paraben includes a member selected from thegroup consisting of parahydroxybenzoate, an ester of parahydroxybenzoicacid, and propyl paraben.

In some embodiments, the analyte preservative includes a proteaseinhibitor.

In some embodiments, the protease inhibitor includes a member selectedfrom the group consisting of serine protease inhibitors, metalloproteaseinhibitors, aminopeptidase inhibitors, cysteine peptidase inhibitor, andaspartyl protease inhibitors.

In some embodiments, the analyte preservative includes an acid.

In some embodiments, the analyte preservative includes at least onemember selected from the group consisting of sorbic acid and citricacid.

In some embodiments, the preservative includes at least one bacteriapreservative.

In some embodiments, the bacteria preservative reduces bacterial growthand multiplication.

In some embodiments, the bacteria preservative includes a bactericidalor bacteriostatic preservative.

In some embodiments, the bacteria preservative includes a preservativefor at least one bacterium associated with at least one GI disorder.

In some embodiments, the bacteria preservative includes a memberselected from the group consisting of sorbic acid, citric acid, propylparaben, nisin, dimethyl dicarbonate, ethylenediaminetetraacetic acid(EDTA), sodium azide, hydroxyurea, fusidic acid, diazolidinyl urea,imidazolidinyl urea, salicylic acid, barium and nickle chloride,metallic copper, thimerosal, 2-phenoxyethanol, and ProClin.

In some embodiments, the bacteria preservative is sorbic acid,thimerosal, 2-phenoxyethanol, diazolinidyl urea, or imidazolinidyl urea.

In some embodiments, the absorbent member includes at least one analytepreservative in addition to the at least one bacteria preservative.

In some embodiments, the analyte preservative is a nucleic acidpreservative.

In some embodiments, the nucleic acid preservative is a DNAse inhibitoror an RNase inhibitor.

In some embodiments, the sampling system includes a plurality ofdifferent preservatives.

In some embodiments, the sampling system includes: a first absorbentmember; and a second absorbent member different from the first absorbentmember. The sampling system is configured so that fluid that flows fromthe exterior of the ingestible device to the interior of the ingestibledevice enters the first absorbent member. The sampling system isconfigured to allow fluid to flow from the first absorbent member to thesecond absorbent member.

In some embodiments, the sampling system further includes a cell filterbetween the first and second absorbent members.

In some embodiments, the sampling system further includes a cell filter.

In some embodiments, the fluid includes a GI fluid.

In some embodiments, the sampling system is configured to fit within aningestible device.

In some embodiments, the sampling system is configured to fit within aningestible device that does not include analytical instrumentation.

In one general aspect, the disclosure provides a method that includescollecting a sample into a sampling system which includes an absorbentmember and at least one preservative at least partially absorbed in theabsorbent member.

In some embodiments, the sampling system is a sampling system asdisclosed herein.

In some aspects, an ingestible device is provided herein. The ingestibledevice includes a housing defined by a first end, a second endsubstantially opposite from the first end, and a wall extendinglongitudinally from the first end to the second end; a first opening inthe wall of the housing; a second opening in the first end of thehousing, the second opening being oriented substantially perpendicularto the first opening; and a curved chamber connecting the first openingand the second opening, wherein at least a portion of the curved chamberforms a sampling chamber within the ingestible device.

In at least some embodiments, the sampling chamber is configured to holda sample obtained from a gastrointestinal (GI) tract of a body.

In at least some embodiments, the ingestible device further comprising amechanical actuator coupled to at least one moveable valve having atleast an open position and a closed position, wherein the at least onemoveable valve in the closed position prevents fluid from entering thesampling chamber and prevents the sample from exiting the samplingchamber.

In at least some embodiments, the ingestible device further comprising amicroprocessor configured to control the mechanical actuator to move theat least one moveable valve into the open position.

In at least some embodiments, the at least one moveable value comprises:a first moveable valve coupled to the mechanical actuator, wherein thefirst movable valve in the closed position prevents fluid from enteringthe sampling chamber via the first opening and prevents the sample fromexiting the sampling chamber via the first opening; and a second movablevalve coupled to the mechanical actuator, wherein the second moveablevalve in the closed position prevents fluid from entering the samplingchamber via the second opening and prevents the sample from exiting thesampling chamber via the second opening.

In at least some embodiments, the first moveable valve in the closedposition is contained in a first portion of the curved chamber locatedbetween the sampling chamber and the first opening; the second moveablevalve in the closed position is contained in a second portion of thecurved chamber located between the sampling chamber and the secondopening; and the first portion of the curved chamber, the second portionof the curved chamber, and the mechanical actuator are oriented in asubstantially straight line, such that the mechanical actuator isconfigured to simultaneously move the first moveable valve and thesecond moveable valve.

In at least some embodiments, the first movable valve and the secondmoveable valve are rotary valves, and the mechanical actuator isconfigured to simultaneously rotate the first movable valve and thesecond moveable valve between the closed position and the open position.

In at least some embodiments, the first moveable valve and the secondmoveable valve are pin valves, the mechanical actuator is configured tosimultaneously move the first moveable valve and the second moveablevalve linearly, and the mechanical actuator comprises at least one of(1) a linear actuator and (2) a rotary actuator coupled to a lead screw.

In at least some embodiments, the ingestible further comprises anelement positioned within the curved chamber proximate to the secondopening that restricts fluid from entering the curved chamber via thesecond opening, the element comprising at least one of a hydrophobicmaterial, an air permeable membrane and a one-way valve.

In at least some embodiments, the ingestible device comprises a sensorwithin or proximate to the sampling chamber for detecting at least oneof (1) a property of the sample, and (2) a result of an assay techniqueapplied to the sample.

In at least some embodiments, the ingestible device comprises at leastone sub-chamber connected to the curved chamber, the at least onesub-chamber being configured to hold a sample obtained from agastrointestinal (GI) tract of a body and isolate the sample from thesampling chamber.

In at least some embodiments, the ingestible device further comprises aplurality of sub-chambers connected to the curved chamber, each of theplurality of sub-chambers being configured to obtain a sample from agastrointestinal (GI) tract of a body at a different time.

In at least some embodiments, the ingestible device further comprises aplurality of sub-chambers connected to the curved chamber, each of theplurality of sub-chambers being configured to obtain a sample from agastrointestinal (GI) tract of a body from a different portion of thegastrointestinal (GI) tract.

In some aspects, another ingestible device is provided herein. Theingestible devices includes a housing defined by a first end, a secondend substantially opposite from the first end, a wall extendinglongitudinally from the first end to the second end, and an opening; asampling chamber within the housing, wherein the sampling chambercontains an absorptive material; an inlet port connecting the opening inthe housing to the sampling chamber; a single use sealing devicepositioned within the inlet port that seals the inlet port; and aheating element proximate to the single use sealing device, wherein: theheating element is configured to apply heat to the single use sealingdevice to unseal the inlet port and open the sampling chamber, and atleast a portion of the absorptive material proximate to the inlet portis configured to expand when in contact with a sample and reseal theinlet port.

In at least some embodiments, the ingestible device comprises amicroprocessor configured to control the heating element to generateheat.

In at least some embodiments, the ingestible device comprises a barrierwithin the sampling chamber positioned between the absorptive materialand the inlet port, the barrier covering a surface of the absorptivematerial.

In at least some embodiments, the barrier separates the absorptivematerial from a remaining portion of the sampling chamber including theinlet port.

In at least some embodiments, the barrier comprises: a first portionproximal to the inlet port and comprising a flexible membrane, and asecond portion adjacent to the first portion and comprising a rigidmaterial.

In at least some embodiments, at least a portion of the absorptivematerial adjacent to the flexible membrane absorbs at least a portion ofthe sample and expands, causing the flexible membrane to reseal theinlet port.

In at least some embodiments, a portion of the sampling chamber betweenthe second portion of the barrier and a wall of the sampling chamberforms a testing area, and a sensor within or proximate to the samplingchamber is configured to detect at least one of (1) a property of thesample within the testing area, and (2) a result of an assay techniqueapplied to the sample within the testing area.

In at least some embodiments, the first portion of the barrier and thesecond portion of the barrier do not allow the sample to pass throughthe barrier and contact the absorptive material.

In at least some embodiments, the barrier comprises a third portionadjacent to the second portion, the third portion comprising asemi-permeable membrane.

In at least some embodiments, the semi-permeable membrane allows atleast a portion of the sample to pass through the semi-permeablemembrane and contact the absorptive material.

In at least some embodiments, the semi-permeable membrane is rigid.

In at least some embodiments, the single use sealing device is abreakable membrane.

In at least some embodiments, the single use sealing device is a plug.

In at least some embodiments, the plug comprises a material with amelting point between 38 degrees Celsius and 80 degrees Celsius, theheating element comprises an electrically conductive element warmed byohmic heating, and the heating element heats the plug to at least themelting point.

In at least some embodiments, the inlet port has a cross-sectional arealess than 50 square millimeters.

In at least some embodiments, the ingestible device comprises at leastone sub-chamber connected to the sampling chamber, the at least onesub-chamber being configured to hold a second sample obtained from agastrointestinal (GI) tract of a body and isolate the second sample fromthe sampling chamber.

In at least some embodiments, the ingestible device comprises aplurality of sub-chambers connected to the sampling chamber, each of theplurality of sub-chambers being configured to obtain a different samplefrom a gastrointestinal (GI) tract of a body at a different time.

In at least some embodiments, the ingestible device comprises aplurality of sub-chambers connected to the sampling chamber, each of theplurality of sub-chambers being configured to obtain a different samplefrom a gastrointestinal (GI) tract of a body from a different portion ofthe gastrointestinal (GI) tract.

In some aspects, another ingestible device is provided herein. Theingestible device includes a housing defined by a first end, a secondend substantially opposite from the first end, a wall extendinglongitudinally from the first end to the second end, and an opening; asampling chamber within the housing having an entry port and an exitport on an opposite end of the sampling chamber from the entry port,wherein the exit port is configured to allow gas to exit the chamber andprevent at least a portion of a sample from exiting the chamber; aninlet region connecting the opening in the housing to the entry port ofthe sampling chamber; and a moveable valve positioned to open and closethe inlet region, wherein: the moveable valve in an open position allowsthe sample to enter the sampling chamber; and the moveable valve in aclosed position prevents the sample from entering the sampling chamber.

In at least some embodiments, the ingestible device comprises amechanical actuator coupled to the moveable valve; and a microprocessorconfigured to control the mechanical actuator to move the moveable valveto the open position.

In at least some embodiments, the moveable valve is a pin valve, themechanical actuator comprises at least one of (1) a linear actuator and(2) a rotating actuator coupled to a lead screw, and the pin valve moveslinearly to switch between the open position and the closed position.

In at least some embodiments, the moveable valve is a rotary valve, themechanical actuator is configured to rotate the rotary valve, and therotary valve rotates to switch between the open position and the closedposition.

In at least some embodiments, the mechanical actuator comprises at leastone of (1) a linear actuator and (2) a rotating actuator coupled to alead screw, and the moveable valve comprises a flexible diaphragm thatmoves from the open position to the closed position by using themechanical actuator to apply pressure across a first surface of theflexible diaphragm.

In at least some embodiments, the ingestible devices comprise a springmechanism positioned proximate to the flexible diaphragm, wherein thespring mechanism applies a counter-pressure across a second surface ofthe flexible diaphragm that is opposite the first surface, such that theflexible diaphragm is in the open position when the mechanical actuatordoes not apply pressure across the first surface.

In at least some embodiments, the exit port comprises a gas permeablemembrane to allow the gas to exit the sampling chamber.

In at least some embodiments, the exit port comprises a one-way valveconfigured to allow gas to exit the sampling chamber and prevent gasfrom re-entering the sampling chamber.

In at least some embodiments, the exit port is connected to an outletport on the housing, the outlet port comprising at least one of a gaspermeable membrane, a one-way valve, and a hydrophobic channel.

In at least some embodiments, the ingestible device includes ahydrophilic sponge within the sampling chamber that is configured toabsorb the sample.

In at least some embodiments, the ingestible device includes a sensorwithin or proximate to the sampling chamber for detecting at least oneof (1) a property of the sample, and (2) a result of an assay techniqueapplied to the sample.

In at least some embodiments, the exit port is connected to a volumewithin the ingestible device, the volume being located outside of thesampling chamber and containing gas.

In at least some embodiments, the exit port is connected to a sealedvacuum chamber with an internal pressure lower than the pressurecontained within at least one of the inlet region and the samplingchamber, the sealed vacuum chamber capable of being unsealed, therebyreducing the pressure in the sampling chamber and drawing the sampleinto the sampling chamber.

In at least some embodiments, moving the moveable valve from the closedposition to the open position causes a volume of the inlet region toincrease.

In at least some embodiments, moving the moveable valve from the openposition to the closed position causes a volume of the inlet region todecrease.

In at least some embodiments, the ingestible device comprises at leastone sub-chamber connected to the inlet region, the at least onesub-chamber being configured to hold a second sample obtained from agastrointestinal (GI) tract of a body and isolate the second sample fromthe sampling chamber.

In at least some embodiments, the ingestible device comprises aplurality of sub-chambers connected to the inlet region, each of theplurality of sub-chambers being configured to obtain a different samplefrom a gastrointestinal (GI) tract of a body at a different time.

In at least some embodiments, the ingestible device comprises aplurality of sub-chambers connected to the inlet region, each of theplurality of sub-chambers being configured to obtain a different samplefrom a gastrointestinal (GI) tract of a body from a different portion ofthe gastrointestinal (GI) tract.

In some aspects, another ingestible device is provided herein. Theingestible device includes a housing defined by a first end, a secondend substantially opposite from the first end, a wall extendinglongitudinally from the first end to the second end, and an opening; asampling chamber within the housing having an entry port; an inletregion connecting the opening in the housing to the entry port of thesampling chamber; and a moveable pump comprising a first portion that isshaped to fit within the opening and a second portion that is shaped tofit within the inlet region; and a mechanical actuator configured tomove the moveable pump to an open position and a fully closed position,wherein: the moveable pump in the open position positions the firstportion of the moveable pump at a distance away from the opening,allowing a sample to enter the inlet region via the opening; and themoveable pump in the fully closed position positions the first portionof the moveable pump within the opening and positions the second portionof the moveable pump adjacent to the entry port, preventing the samplefrom exiting the inlet region via the opening and the entry port.

In at least some embodiments, the mechanical actuator is furtherconfigured to move the moveable pump to a partially closed position,wherein the moveable pump in the partially closed position positions asurface of the first portion of the moveable pump adjacent to theopening, thereby sealing off the opening such that the sample isprevented from exiting the inlet region via the opening.

In at least some embodiments, the partially closed position positionsthe second portion to be away from the entry port, thereby unsealing theentry port such that the sample is allowed to exit the inlet region viathe entry port.

In at least some embodiments, the ingestible device comprises amicroprocessor configured to control the mechanical actuator to move themoveable pump between the fully closed position and the open position.

In at least some embodiments, the mechanical actuator comprises at leastone of (1) a linear actuator and (2) a rotating actuator coupled to alead screw, and the mechanical actuator is usable to move the moveablepump linearly between the fully closed position and the open position.

In at least some embodiments, the ingestible device comprises an exitport on an opposite end of the sampling chamber from the entry port,wherein the exit port is configured to allow gas to exit the chamber andprevent at least a portion of a sample from exiting the chamber.

In at least some embodiments, the exit port comprises a gas permeablemembrane to allow the gas to exit the sampling chamber.

In at least some embodiments, the exit port comprises a one-way valveconfigured to allow gas to exit the sampling chamber and prevent gasfrom re-entering the sampling chamber.

In at least some embodiments, the exit port is connected to an outletport on the housing, the outlet port comprising at least one of a gaspermeable membrane, a one-way valve, and a hydrophobic channel.

In at least some embodiments, the exit port is connected to a volumewithin the ingestible device, the volume being located outside of thesampling chamber and containing gas.

In at least some embodiments, the ingestible device comprises ahydrophilic sponge within the sampling chamber that is configured toabsorb the sample.

In at least some embodiments, the ingestible device comprises a sensorwithin or proximate to the sampling chamber for detecting at least oneof (1) a property of the sample, and (2) a result of an assay techniqueapplied to the sample.

In at least some embodiments, the ingestible device comprises at leastone sub-chamber connected to the inlet region, the at least onesub-chamber being configured to hold a second sample obtained from agastrointestinal (GI) tract of a body and isolate the second sample fromthe sampling chamber.

In at least some embodiments, the ingestible device comprises aplurality of sub-chambers connected to the inlet region, each of theplurality of sub-chambers being configured to obtain a different samplefrom a gastrointestinal (GI) tract of a body at a different time.

In at least some embodiments, the ingestible device comprises aplurality of sub-chambers connected to the inlet region, each of theplurality of sub-chambers being configured to obtain a different samplefrom a gastrointestinal (GI) tract of a body from a different portion ofthe gastrointestinal (GI) tract.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an illustrative embodiment of an ingestible device withmultiple openings in the housing.

FIG. 2 shows another illustrative embodiment of an ingestible device,including various modifications that may be made to the ingestibledevice of FIG. 1.

FIG. 3 shows an illustrative valve design that may be used to obtain asample with an ingestible device.

FIGS. 4 and 5 illustrate how the valve in FIG. 3 may be operated inorder to obtain a sample.

FIG. 6 shows an illustrative embodiment of an ingestible device with asampling chamber that includes an exit port.

FIG. 7 shows different illustrative valve designs that may beincorporated into an ingestible device.

FIG. 8 shows an illustrative sampling chamber that may be incorporatedinto an ingestible device.

FIG. 9 shows an illustrative pumping mechanism that may be incorporatedinto an ingestible device.

FIG. 10 shows a highly schematic representation of an ingestible device.

FIG. 11 shows a highly cross-section of an ingestible device including avalve system and a sampling system.

FIG. 12 illustrates a valve system.

FIGS. 13A and 13B illustrate a portion of a two-stage valve system inits first and second stages, respectively.

FIGS. 14A and 14B illustrate a portion of a two-stage valve system inits first and second stages, respectively.

FIGS. 15A and 15B illustrate a portion of a two-stage valve system inits first and second stages, respectively.

FIG. 16 illustrates a more detailed view of an ingestible deviceincluding a valve system and a sampling system.

FIGS. 17A-17C illustrate a portion of a three-stage valve system in itsfirst, second and third stages, respectively.

FIGS. 18A-18C illustrate a portion of a three-stage valve system in itsfirst, second and third stages, respectively.

FIGS. 19A-19C illustrate a portion of a three-stage valve system in itsfirst, second and third stages, respectively.

FIG. 20 illustrates a three-stage valve system in its first stage.

FIG. 21A illustrates a portion of an ingestible device including asampling system and a two-stage valve system in its first stage.

FIG. 21B illustrates a portion of an ingestible device including asampling system and a two-stage valve system in its second stage.

FIG. 22 illustrates an ingestible device including a sampling system anda two-stage valve system in its first stage.

FIG. 23 illustrates an ingestible device including a sampling system anda portion of a three-stage valve system in third stage.

FIG. 24 illustrates an ingestible device including a sampling system anda three-stage valve system in third first stage.

FIG. 25 is a highly schematic illustrate of an ingestible device.

FIG. 26 is an exploded view of an ingestible device.

FIG. 27 illustrates a portion of an ingestible device with a port in anopen position exposed to the exterior of device.

FIG. 28 illustrates a portion of an ingestible device with a port in afirst position in fluid communication with a first incubation chamber.

FIG. 29 illustrates a member forming part of a set of five incubationchambers suitable for an ingestible device.

FIG. 30 illustrates a partial cross-sectional view of optics in aningestible device.

FIG. 31 illustrates components of the optics and flow chamber systems inan ingestible device.

FIG. 32 shows a partial view of an ingestible device

FIGS. 33A-33C show illustrate operation of ingestible device 5010.

FIG. 34 illustrates an exploded view of the components of ingestibledevice.

FIG. 35 depicts a dilution series.

FIG. 36 shows ELISA data.

FIG. 37 shows ELISA data.

FIG. 38 shows ELISA data.

FIG. 39 shows ELISA data.

FIG. 40 shows ELISA data.

FIG. 41 shows ELISA data.

FIG. 42 shows ELISA data.

FIG. 43 shows ELISA data.

FIG. 44 shows ELISA data.

FIG. 45 shows ELISA data.

FIG. 46 shows ELISA data.

FIG. 47 shows ELISA data.

FIG. 48 shows ELISA data.

FIG. 49 shows ELISA data.

FIG. 50 shows ELISA data.

FIG. 51 shows ELISA data.

FIG. 52 shows ELISA data.

FIG. 53 shows ELISA data.

FIG. 54 shows ELISA data.

FIG. 55 shows data on bacteria amount as a function of time.

FIG. 56A-56C show bacteria recovery data.

FIG. 57 shows data on fluid absorption.

FIG. 58 shows data on inhibition/preservation of bacterial population.

FIGS. 59A and 59B show data on reduction of viability of bacteria.

FIG. 60 shows data on reduction of viability of bacteria.

DETAILED DESCRIPTION

To provide an overall understanding of the disclosure, certainillustrative embodiments will now be described, including varioussystems and methods for obtaining samples using ingestible devices. Inparticular, techniques are described that allow an ingestible device toobtain a sample from within a gastrointestinal (GI) tract. These samplesmay include any of the fluids, solids, particulates, or other substancesfound within the GI tract. However, it will be understood by one ofordinary skill in the art that the systems and methods described hereinmay be adapted and modified as is appropriate for the applications beingaddressed, and that the systems and methods described herein may beemployed in other suitable applications, and that such other additionsand modifications will not depart from the scope of the presentdisclosure. Generally, the ingestible devices described herein maycomprise actuators, sensors, valves, chambers, logic devices, telemetrysystems, microcontrollers or other devices and processors that may beconfigured using a combination of hardware, firmware, and software tocarry out one or more of the methods described herein.

FIG. 1 illustrates an example ingestible device 100 with multipleopenings in the housing. The ingestible device 100 has an outer housingwith a first end 102A, a second end 102B, and a wall 104 extendinglongitudinally from the first end 102A to the second end 102B.Ingestible device 100 has a first opening 106 in the housing, which isconnected to a second opening 108 in the housing. The first opening 106of the ingestible device 100 is oriented substantially perpendicular tothe second opening 108, and the connection between the first opening 106and the second opening 108 forms a curved chamber 110 within theingestible device 100.

The overall shape of the ingestible device 100, or any of the otheringestible devices discussed in this disclosure, may be similar to anelongated pill or capsule. This may make the ingestible device 100 easyto consume, and allow it to travel easily through the GI tract. As usedherein, the term “gastrointestinal tract” or “GI tract” refers to allportions of an organ system responsible for consuming and digestingfoodstuffs, absorbing nutrients, and expelling waste. This includesorifices and organs such as the mouth, throat, esophagus, stomach, smallintestine, large intestine, rectum, anus, and the like, as well as thevarious passageways and sphincters connecting the aforementioned parts.In certain portions of the GI tract, such as the stomach, the ingestibledevice 100 may be free to move or rotate in any direction. In otherportions of the GI tract, the movement of the ingestible device 100 maybe restricted. For example, in the relatively narrow confines of thesmall intestine, the walls of the small intestine may squeeze down onthe ingestible device, forcing the ingestible device 100 to orientitself longitudinally along the length of the small intestine. In thiscase, the walls of the small intestine wrap around the longitudinallyextending wall 104 of the ingestible device 100, and the ingestibledevice 100 travels through the small intestine with one of the ends 102Aor 102B in front.

For illustrative purposes, the ingestible device 100 of FIG. 1 shows thefirst opening 106 located in a portion of the wall 104 and orientedradially, and the second opening 108 located near the first end 102A andoriented longitudinally. However, in some embodiments, the exactlocation and orientation of the first opening 106 and the second opening108 may be different from that shown in FIG. 1. During transit throughthe GI Tract, natural contractions within the small intestine may applypressure radially to different portions of the wall 104 of theingestible device 100, which may force solids or fluids into the firstopening 106. As new material (e.g., fluid and solid particulates fromthe small intestine or other portions of the GI tract) enters the curvedchamber 110 through the first opening 106, older material alreadylocated in the curved chamber 110 may be naturally forced out of thecurved chamber 110 through the second opening 108.

In some embodiments, a portion of the curved chamber 110 may be used asa sampling chamber, which may hold samples obtained from the GI tract.In some embodiments the curved chamber 110 is subdivided intosub-chambers, each of which may be separated by a series of one or morevalves or interlocks. For example, sub-chambers may be used to retainmultiple samples within different portions of the curved chamber 110. Insome embodiments, the curved chamber 110 is connected to other chamberswithin the ingestible device 100, or other openings located on thehousing of the ingestible device 100. This may allow new samples to beacquired in the curved chamber 110 while older samples of interest arestill stored within the ingestible device 100. In some embodiments, theingestible device 100 is equipped with sensors to detect the propertiesa sample contained in the sampling chamber, or the results of an assaytechnique applied to the sample. In some embodiments, the ingestibledevice 100 is configured to obtain and retain a sample within thesampling chamber, which may be retrieved at a later time.

In some embodiments, the first opening 106, the second opening 108, orthe curved chamber 110 include one or more of a hydrophilic orhydrophobic material, a sponge, a valve, or an air permeable membrane.For example, a one-way valve may prevent material from entering thecurved chamber 110 through the second opening 108. As an alternateexample, placing an air permeable membrane within the curved chamber 110near the second opening 108 may allow unwanted gasses and air bubbles topass through the air permeable membrane and exit the curved chamber 110,while solid or liquid samples may be prevented from passing through theair permeable membrane, and are retained within the curved chamber 110.The air permeable membrane may also prevent solid or liquid samples fromentering the curved chamber 110 through the second opening 108.

The use of a hydrophilic material or sponge may allow samples to beretained within the curved chamber 110, and may reduce the amount ofpressure needed for fluid to enter through the first opening 106 anddislodge air or gas in the curved chamber 110. Examples of hydrophilicmaterials that may be incorporated into the ingestible device 100include hydrophilic polymers such as polyvinyl alcohol, polyvinylpyrrolidone, and the like. Similarly, materials that have undergonevarious types of treatments, such as plasma treatments, may havesuitable hydrophilic properties, and may be incorporated into theinvestible device 100. Sponges may be made of any suitable material orcombination of materials, such as fibers of cotton, rayon, glass,polyester, polyethylene, polyurethane, and the like. Sponges generallymay be made from commercially available materials, such as thoseproduced by Porex®.

As discussed in more detail below, in some embodiments, the sponges maybe treated in order to change their absorbency or to help preservesamples.

In some embodiments, the sponges may be cut or abraded to change theirabsorbency or other physical properties.

Hydrophobic materials located near the second opening 108 may repelliquids, discouraging liquid samples from entering or exiting the curvedchamber 110 through the second opening 108. This may serve a similarfunction as an air permeable membrane. Examples of hydrophobic materialswhich may be incorporated into the ingestible device 100 includepolycarbonate, acrylics, fluorocarbons, styrenes, certain forms ofvinyl, and the like.

The various materials listed above are provided as examples, and are notlimiting. In practice, any type of suitable hydrophilic, hydrophobic, orsample preserving material may be used in the ingestible device 100, andthe teachings discussed in relation to ingestible device 100 may beincorporated into any of the other ingestible devices described in thisdisclosure. Various methods for taking samples, controlling the movementof samples, or removing unwanted gasses, are discussed in detail inrelation to FIGS. 2-9, and any of the various structures or techniquesdescribed in connection with FIGS. 2-9 may be incorporated into theingestible device 100.

FIG. 2 illustrates an example ingestible device 200 with multipleopenings in the housing and various modifications that may be made tothe ingestible device 100. Similar to the ingestible device 100, theingestible device 200 has an outer housing with a first end 202A, asecond end 202B, and a wall 204 extending longitudinally from the firstend 202A to the second end 202B. Also similar to the ingestible device100, the ingestible device 200 has a first opening 206 in the housing,which is connected to a second opening 208 in the housing. Theconnection between the first opening 206 and the second opening 208forms a curved chamber 210 within the ingestible device 200.

In the ingestible device 200, a portion of the curved chamber 210 formsa sampling chamber 212. In some embodiments, the ingestible device 200may include a sensor (not shown) within or proximate to the samplingchamber. This sensor may be used to detect a property of the sample. Insome embodiments, an assay technique is applied to a sample within thesampling chamber, and the sensor may be used to detect the results ofthe assay technique. A first valve 214 is located between the firstopening 206 and the sampling chamber 212. Similarly, a second valve 216is located between the second opening 208 and the sampling chamber 212.In some embodiments, the valves 214 and 216 prevent a fluid fromentering or exiting the sampling chamber 212, or may be used to isolatea sample within the sampling chamber 212.

The ingestible device 200 includes a mechanical actuator 218 coupled tothe valves 214 and 216. In some embodiments, the mechanical actuator 218is used to move one or both of the valves 214 and 216 between an openand a closed position. In some embodiments, the mechanical actuator 218is controlled by a microcontroller, microprocessor, or other circuitryinside the ingestible device 200. In an open position, the first valve214 may allow a sample to pass in and out of the sampling chamber 212through the portion of the curved chamber 210 connected to the firstopening 206. Similarly, in an open position, the second valve 216 mayallow a sample to pass in and out of the sampling chamber 212 from theportion of the curved chamber 210 connected to the second opening 208.When the valves 214 and 216 are in the closed positions, they may notallow a sample to pass into or out of the sampling chamber 212.

In some embodiments, the valves 214 and 216 are rotary valves, pinvalves, flap valves, butterfly valves, ball valves, plug valves, or anyother suitable type of one-way or two-way valves, and may be the same ordifferent types of valves. In some embodiments, one or both of thevalves 214 and 216 are automatic valves that reseal themselves after asample has been obtained, similar to the osmotic valve mechanismdiscussed in relation to FIG. 3. In some embodiments, one or both of thevalves 214 and 216 include a pumping mechanism, such as the pumpingmechanism discussed in relation to FIG. 9. For illustrative purposes,the ingestible device 200 is depicted with both of the valves 214 and216 as moveable two-way valves coupled to the mechanical actuator 218.However, in some embodiments, the mechanical actuator 218 is coupled toonly one of the valves, and the other valve may be replaced with apassive one-way valve. For example, the mechanical actuator 218 may becoupled to only the first valve 214, and the second valve 216 may bereplaced with a passive one-way valve that allows gases, fluids, orsolids to exit the sampling chamber 212 through the portion of thecurved chamber 210 connected to the second opening 208. This mayrestrict fluid from entering the sampling chamber 212 from the secondopening 208, but allow unwanted material to be removed from the samplingchamber 212 as the sample is obtained.

In some embodiments, the ingestible device 200 may be able to detect theapproximate location of the ingestible device 200 within the GI tract.For example, it may be possible to use various combinations of lightemitting diodes and sensors positioned along the ingestible device 200to determine whether the device is in the stomach, small intestine, orlarge intestine. Methods for determining the location of an ingestibledevice within a gastrointestinal tract are described in greater detailin PCT Application No. PCT/US15/52500 filed 25 Sep. 2015, which ishereby incorporated by reference herein in its entirety. In theseembodiments, the ingestible device 200 may be configured to use themechanical actuator 218 to move the valves 214 and 216 into an openposition in response to determining that the ingestible device 200 hasreached a predetermined location within the GI tract. For example, amicrocontroller on board the ingestible device 200 may be configured toopen the valves 214 and 216 only when the ingestible device 200 iswithin the small intestine, thereby obtaining a sample from within thesmall intestine.

For illustrative purposes, the ingestible device 200 is depicted withthe mechanical actuator 218, the first valve 214, and the second valve216 oriented in a substantially straight line, with a single shaft 220being used to couple the mechanical actuator 218 to the valves 214 and216. However, in some embodiments, the orientation and/or positioning ofthe valves 214 and 216 relative to the position of the mechanicalactuator 218 may be different than that shown, and the coupling of themechanical actuator 218 to the valves 214 and 216 may also be different.In some embodiments, the mechanical actuator 218 simultaneously movesthe valves 214 and 216. For example, in some embodiments the valves 214and 216 are rotary valves, and they may be simultaneously opened andclosed by rotating the shaft 220 that extends from the mechanicalactuator 218 along the length of the ingestible device 200. As analternate example, the valves 214 and 216 may be pin valves, and thepins may be attached to the shaft 220 that extends from the mechanicalactuator 218 along the length of the ingestible device 200. In thiscase, the mechanical actuator 218 may open and close the valves bymoving the shaft 220 linearly. This may be accomplished either byconfiguring mechanical actuator 218 to be a linear actuator, such as asolenoid. Alternately, the mechanical actuator 218 may be a rotaryactuator, and the rotation may be converted into a linear motion. Oneskilled in the art will understand that this may be done any number ofways, for example, by coupling the mechanical actuator 218 to a ballscrew mechanism, a threaded lead nut and lead screw mechanism, a rackand pinion mechanism, or the like.

In some embodiments, the ingestible device 200 does not include thesecond valve 216 at all. In this case, fluids and solids containedwithin the sampling chamber 212 may be free to exit through the secondopening 208. Alternately, the second valve 216 near the second opening208 may be replaced by an air-permeable membrane, which may allow gassesand unwanted air bubbles to exit the sampling chamber 212 through thesecond opening 208, while still retaining fluids and/or solids withinthe sampling chamber 212. Alternately, the second valve 216 near thesecond opening 208 may be replaced with a hydrophobic material. Similarto an air permeable membrane, an appropriately positioned hydrophobicmaterial may be used to line the walls of the curved chamber 210proximate to the second opening 208, which may allow gasses or unwantedair bubbles to exit the sampling chamber 212 through the second opening208, while restricting some fluids from entering or exiting the samplingchamber 212 through the second opening 208. In some embodiments, one ormore of the above described mechanisms may be combined in the sameingestible device. For example, the ingestible device 200 may implementthe second valve 216 as a two-way valve, and also have hydrophobicmaterial and an air-permeable membrane located near the second opening208.

In some embodiments, the curved chamber 210 is connected to one or moresub-chambers (not shown). Each of these sub-chambers may be configuredto hold one or more samples, and isolate the samples from both thesampling chamber 212, and the other sub-chambers. For example, eachsub-chamber may be connected to the curved chamber 210 through a one-wayvalve, allowing samples to enter the sub-chamber from the curved chamber210, but preventing the obtained samples from exiting the sub-chamberand re-entering either the curved chamber 210 or the sampling chamber212. In general, any type of valve or other suitable mechanism may beused to isolate samples contained in the sub-chambers. In someembodiments, the ingestible device 200 distributes different samplesinto different sub-chambers at different times, or from differentlocations of the GI tract. For example, the ingestible device 200 mayobtain a sample from the duodenum and distribute it into a firstsub-chamber, and the ingestible device 200 may later obtain a samplefrom the ileum and distribute it into a second sub-chamber. In someembodiments, different types of assay techniques or diagnostics areapplied to some of the samples contained in the different sub-chambers.

FIG. 3 illustrates an example of an osmotic valve mechanism 300, whichmay be incorporated into an ingestible device in order to obtainsamples. The osmotic valve mechanism 300 may be used in an ingestibledevice that features a first end, a second end, and a wall extendinglongitudinally between the first end and the second end, similar to theshape of the ingestible devices 100 (FIG. 1) and 200 (FIG. 2).

The osmotic valve mechanism 300 includes an inlet port 302, which isconnected to a sampling chamber 304. In some embodiments, the inlet port302 connects sampling chamber 304 directly or indirectly to an openingin the housing of an ingestible device.

The initial state of the osmotic valve mechanism 300 is shown in diagram300A. As shown in diagram 300A, the inlet port 302 of the osmotic valvemechanism 300 is sealed using a single use sealing device 306 positionedwithin the inlet port 302. The single use sealing device 306 ispositioned adjacent to a heating element 308. When it is time for theosmotic valve mechanism 300 to be opened (which may be determined by alocalization mechanism that determines the ingestible device is locatedin a desirable portion of the GI tract), the heating element 308 appliesheat to the sealing device 306, causing the sealing device 306 to deformand unseal the inlet port 302.

In some embodiments, the sealing device 306 may be a plug made out of amaterial that is meltable, deformable, and/or destroyable through theuse of the heating element 308, such as wax. For example, in oneembodiment, the heating element 308 may be a resistive heater thatundergoes ohmic heating as an electrical current is passed through it,and the sealing device 306 is a wax plug. In some embodiments, the typeof wax used to form the wax plug has a melting point between 38 degreesand 80 degrees Celsius, which is above the ambient temperature of ahuman body, but which may be easily achieved using the heating element308. Some embodiments of the osmotic valve mechanism 300 may use asealing device 306 that is melted or deformed at temperatures outside ofthe range described above, but practical considerations may be made toensure that the osmotic valve mechanism 300 does not cause unwanteddamage or burning to the GI tract. In some embodiments, a microprocessoris configured to control the heating element 308, causing it to generateheat. For example, the microprocessor may be configured to activate theheating element 308 once the ingestible device reaches a particularlocation within the GI tract. An example mechanism for unsealing theinlet port 302 is described in greater detail in relation to FIGS. 4 and5. Although FIGS. 3, 4, and 5 depict the sealing device 306 as a type ofplug, any type of suitable sealing device may be used. For example, insome embodiments, the sealing device includes a breakable membrane,which may be destroyed when heat is applied to the membrane. In someembodiments, the osmotic valve mechanism 300 does not include a heatingelement 308, and the sealing device 306 is melted, deformed, destroyed,or dislodged from the inlet port 302 by a mechanical actuator, orthrough electromagnetic fields. For example, the sealing device 306 maybe a membrane that will rupture when a sufficiently large electricalcurrent or magnetic field is applied to the membrane.

Inside the sampling chamber 304 of the osmotic valve mechanism 300 is anabsorptive material 310, and at least a portion of the absorptivematerial 310 is located near the inlet port 302. The absorptive material310 may include any suitable sponge material or hydrophilic material,such as any of the materials described in relation to FIG. 1. Theportion of the absorptive material 310 located near the inlet port 302may have a tendency to expand when it comes into contact with fluids.The osmotic valve mechanism 300 has a barrier 312 inside the samplingchamber 304, which is divided into three portions. The first portion ofthe barrier 312 is a flexible membrane 314, the second portion of thebarrier 312 adjacent to the flexible membrane 314 is a rigid portion316, and the third portion of the barrier 312 adjacent to the rigidportion 316 is a semi-permeable membrane 318.

The barrier 312 within the sampling chamber 304 is positioned betweenthe inlet port 302 and the absorptive material 310, covering a surfaceof the absorptive material 310. When the inlet port 302 is unsealed, asample (e.g., a fluid sample containing solid particulates taken fromthe GI tract) enters the sampling chamber 304 through the inlet port302, and begins to fill the sampling chamber 304. The absorptivematerial 310 may have a natural tendency to expand when it comes intocontact with a fluid sample. However, by covering a surface of theabsorptive material 310, the barrier 312 may allow only certain portionsof absorptive material 310 to expand. The barrier 312 may also directthe flow of a fluid sample as it enters the sampling chamber 304, andallow the fluid sample to come into contact with only certain parts ofthe absorptive material 310.

Diagram 300B shows the osmotic valve mechanism 300 shortly after theinlet port 302 is unsealed. Once the inlet port 302 is unsealed, thesampling chamber 304 may be opened, and a sample may enter the samplingchamber 304 through the inlet port 302. In some embodiments, the samplecannot cross the flexible membrane 314 and contact the absorptivematerial 310. As a result, the flexible membrane 314 may be used toguide the sample as it enters the sampling chamber 304. Similarly, insome embodiments the sample cannot cross the rigid portion 316 of thebarrier 312, and the rigid portion 316 may also be used to guide thesample as it enters the sampling chamber 304. The semi-permeablemembrane 318 allows at least a portion of the sample to pass through thesemi-permeable membrane and contact the absorptive material 310. Thismay allow the sample to be absorbed by the absorptive material 310 afterthe sample has filled the top portion of the sampling chamber 304, whichin turn may cause the absorptive material 310 to begin to expand.

Diagram 300C shows the state of the osmotic valve mechanism 300 afterthe absorptive material 310 has absorbed a portion of the sample. Theportion of the absorptive material 310 under the flexible membrane 314expands when the absorptive material 310 absorbs the sample. As theabsorptive material 310 expands, the flexible membrane 314 is forced upagainst the inlet port 302, effectively sealing the inlet port 302 fromthe sampling chamber 304. In some embodiments, the rigid portion 316prevents the portion of the absorptive material 310 under the rigidportion 316 from expanding. In some embodiments, the semi-permeablemembrane 318 may be rigid, and prevent the portion of the absorptivematerial 310 adjacent to the semi-permeable membrane 318 from expanding.

After the absorptive material 310 expands, causing the inlet port 302 tobe resealed, a portion of the sample may be confined within the samplingchamber 304. Once a sample has been properly confined, it may bepossible to apply a wide range of assay techniques or diagnostics to thesample. In some embodiments, the portion of the sampling chamber 304between the rigid portion 316 and the wall of the sampling chamber formsa testing area. For example, a sensor may be placed within or proximateto the sampling chamber 304 in order to study the portion of the samplecontained within the testing area located above the rigid portion 316.This sensor may be used to study properties of the sample, or it may beused to detect the results of an assay technique applied to the sample.

Diagram 300C is shown for illustrative purposes only, and is notlimiting. In some embodiments, the osmotic valve mechanism 300 does notinclude the barrier 312, or one or more portions of the barrier 312 areexcluded or rearranged within the sampling chamber 304. For example, thelocation of the rigid portion 316 and the semi-permeable membrane 318may be reversed, or the rigid portion 316 may be removed and thesemi-permeable membrane 318 extended so that it connects directly withthe flexible membrane 314. When the osmotic valve mechanism 300 does notinclude a barrier 312 or does not include the flexible membrane 314, aportion of the absorptive material 310 near the inlet port 302 mayexpand and clog the inlet port 302, effectively resealing the inlet port302.

In some embodiments, the material used to form the absorptive material310 expands at a controlled rate, which may ensure that sufficient timehas passed for the sample to enter the sampling chamber 304 and for thesampling chamber 304 to be filled before the inlet port 302 is resealed.This may be particularly useful for embodiments where the osmotic valvemechanism 300 does not include a flexible membrane 314 and/or thesemi-permeable membrane 318. In some embodiments, a portion of theabsorptive material 310 is covered by a dissolvable film or membrane,which may prevent the absorptive material 310 from expanding until asufficient amount of time has passed for the film to dissolve.

In some embodiments, the sampling chamber 304 is connected to one ormore sub-chambers (not shown). Each of these sub-chambers may beconfigured to hold samples, and isolate the samples from both thesampling chamber 304, and the other sub-chambers. For example, eachsub-chamber may be connected to the sampling chamber 304 through aone-way valve, allowing samples to enter the sub-chamber from thesampling chamber, but preventing the obtained samples from exiting thesub-chamber. As an alternate example, each of the sub-chambers mayemploy a sealing device, heating element, and absorptive materialarranged similar to osmotic valve mechanism 300. In these embodiments,each of the sub-chambers may be opened by activating their respectiveheating elements, and may be automatically sealed off from the samplingchamber 304 after a sufficient amount of the sample has been obtained.In general, any type of valve or other suitable mechanism may be used toisolate samples contained in the sub-chambers. In some embodiments,similar to ingestible device 200, an ingestible device employingmultiple sub-chambers in conjunction with the osmotic valve mechanism300 may distribute different samples into different sub-chambers atdifferent times, or from different locations of the GI tract.

It will be understood by one skilled in the art that variations of theosmotic valve mechanism 300 may be combined with any of the otheringestible devices described in this disclosure. For example, in someembodiments of the ingestible device 200 shown and described in relationto FIG. 2, one or both of the valves 214 and 216 may be replaced withcertain embodiments of the osmotic valve mechanism 300. One or both ofthe valves 214 and 216 may include a sealing device that can bedestroyed or deformed (e.g., by the mechanical actuator 218 or through aheating element), and one or both of the valves 214 and 216 may beautomatically resealed by the expansion of absorptive material locatedwithin the sampling chamber 212.

FIGS. 4 and 5 illustrate in detail how some embodiments of the osmoticvalve mechanism 300 (FIG. 3) may be operated in order to obtain asample.

FIG. 4 shows a detailed view of an inlet port 400, which may beincorporated into osmotic valve mechanism 300, prior to being unsealed.The inlet port 400 features an exterior portion 402, which is separatedby a middle portion 404 from an interior portion 406. The middle portion404 of the inlet port 400 contains a sealing device 408, which may bethe same as sealing device 306 shown and described in relation to FIG.3. A heating element 410 is located near the middle portion 404, andadjacent to the sealing device 408. The sides of the inlet port 412A and412B form the shape of the inlet port 400, and may be constructed froman insulating material, such as insulating ceramic, or polymers such aspolyamide-imide, polyphenylene sulfide, polyphenylene oxide, and thelike. For illustrative purposes, the exterior portion 402 of the inletport 400 is depicted as being filled with a sample 414, which may be afluid sample obtained from the GI tract. However, in some embodiments,the inlet port 400 may be operated regardless of whether a sample 414 isactually contained in the exterior portion 402. The exterior portion 402and the interior portion 406 are wider than the middle portion 404. Asloped wall 416 gradually reduces the width of the exterior portion 402,to transition from the wider width of the exterior portion 402 to thenarrower width of the middle portion 404. This configuration may reducethe overall volume of the sealing device 408 (compared to aconfiguration with a wider middle portion 404), and reduce the surfacearea of the sealing device 408 exposed to the sample 414, which mayreduce the amount of heat lost from the sealing device 408 to the sample414. In turn, this may make it easier to raise the temperature of thesealing device 408 using the heating element 410. In some embodiments,the geometry of the inlet port 400 may allow an air pocket (not shown)to form in the exterior portion 402, separating the sealing device 408from fluid contained within the GI tract. This may act as an insulatingbarrier around the sealing device 408, and also make it easier to raisethe temperature of the sealing device 408 using the heating element 410.Moreover, the larger width of the interior portion 406 relative to themiddle portion 404 forms a remnant capture area 418, which may hold theremnants of the sealing device 408 after the inlet port 400 is unsealed.

In some embodiments, the exterior portion 402 of the inlet port 400 maybe connected directly or indirectly to an opening in the housing of aningestible device. In some embodiments, there is nothing to restrict asample from entering the opening, and, at any given time, the exteriorportion 402 of the inlet port 400 may be filled with a fluid sample 414gathered from whatever portion of the GI tract the ingestible device islocated within.

Sealing device 408 prevents the fluid sample 414 contained within theexterior portion 402 of the inlet port 400 from entering the interiorportion 406 of the inlet port 400. For simplicity, FIGS. 4 and 5 depictthe sealing device 408 as a plug, which forms a seal that may be brokenby using a heating element 410. However, in some embodiments the sealingdevice 408 may be any other type of breakable seal or valve used withinthe middle portion 404 to separate the exterior portion 402 of the inletport 400 and the interior portion 406 of the inlet port 400.

In some embodiments, the heating element 410 may be operated by amicrocontroller. For example, the microcontroller may be configured tooperate the heating element 410 and unseal the inlet port 400 when theingestible device is in a certain portion of the GI tract. The sides ofthe inlet port 412A and 412B may be formed from an insulating material,which may shield the ingestible device and the fluid sample 414 from theheat generated by the heating element 410. This may also help to focusthe heat produced by heating element 410 in the direction of the sealingdevice 408, and may reduce the total amount of power to drive theheating element 410 to melt, deform, or destroy the sealing device 408.

In some embodiments, the dimensions of the inlet port 400 are chosensuch that a fluid sample 414 is naturally drawn into the exteriorportion 402, and ultimately through the middle portion 404 into theinterior portion 406, through capillary action. Typically, thecross-section of the exterior portion 402, the middle portion 404, andthe interior portion 406 will be square, circular, or rectangular, butany type of cross-section may be used. The overall cross-sectional areaof the exterior portion 402, the middle portion 404, and the interiorportion 406 of the inlet port 400 is typically less than 50 squaremillimeters given the size constraints of the ingestible device, with0.2 to 2 square millimeters being common. However, the cross-sectionalareas listed above are only examples, and any cross-sectional area maybe chosen in order to better draw in samples from the different portionsof the GI tract. One skilled in the art will understand that the exactshape and dimensions will depend on the physical properties of thesample to be acquired, and some embodiments may use cross-sections otherthan the ones mentioned above.

FIG. 5, shows a detailed view of an inlet port 500, which may beincorporated into osmotic valve mechanism 300, after it has beenunsealed.

After the heating element 510 has heated the sealing device 508sufficiently, the sealing device 508 may deform, melt, or otherwise bedestroyed, effectively unsealing the inlet port 500. Once the inlet port500 is unsealed, the fluid sample 514 is able to flow naturally from theexterior portion 502 of the inlet port 500 to the interior portion 506of the inlet port 500 through the middle portion 504. Similar to theembodiments described in relation to FIG. 4, the sides 512A and 512B ofthe inlet port may be made of an appropriate insulating material, andform the shape of the inlet port 500, the exterior portion 502 with thesloped wall 516, the middle portion 504, and the interior portion 506along with the remnant capture area 518. As the fluid sample 514 entersthe interior portion 506 of the inlet port 500, the natural flow of thefluid sample 514 may carry any of the remnants of the sealing device 508into the remnant capture area 518 located within the interior portion506. In some embodiments, once the melted or deformed remnants of thesealing device 508 cease to be in contact with the heating element 510and instead come into contact with the insulating material that make upthe walls of the remnant capture area 518, the remnants of the sealingdevice 508 re-solidifies or re-forms along the walls of the remnantcapture area 518. As a result, the remnant capture area 518 may providea location for the re-solidified remnants of the sealing device 508 tobe stored, and may prevent the remnants of the sealing device 508 fromimpeding the flow of the sample 514.

In some embodiments, electromagnetic forces are used to attract theremnants of the sealing device 508 to the remnant capture area 518. Forexample, the sealing device (e.g., the sealing device 408) may be madefrom a magnetic material, and an induced or permanent magnetic field maybe used to attract the remnants of the sealing device 508 to the remnantcapture area 518. This magnetic field may be applied after the heatingelement 510 is activated, and until the remnants of the sealing device508 re-solidify or re-form within the remnant capture area 518.

It will be understood that the embodiments described by FIGS. 3, 4, and5, are merely illustrative, and they may be modified and combined withother techniques for drawing in or pumping fluid samples withoutdeparting from the spirit and scope of this disclosure. For example, toencourage samples to be drawn into the sampling chamber 304, thesampling chamber 304 may contain a low-pressure vacuum, and samples maybe forcibly drawn into the sampling chamber 304 when the inlet port 302is unsealed. A similar effect may also be produced by connecting thesampling chamber 304 to a sub-chamber containing a low-pressure vacuum,or by using by using a mechanical actuator to either pump the fluidsamples or to increase the volume of the sampling chamber 304. In someembodiments, the geometry and relative size of the exterior portions 402and 502, the middle portions 404 and 504, and interior portions 406 and506, may be different from those depicted in FIGS. 4 and 5. For example,the different portions 402, 404, 406, 502, 504, and 506 may have auniform width, and the sloped walls 416 and 516 and/or the remnantcapture areas 418 and 518 are not included. As another example, a slopedwall may be used to form the remnant capture areas 418 and 518.

FIG. 6 illustrates another example of an ingestible device 600 with asampling chamber that includes an exit port. Similar to the ingestibledevices 100 and 200, the ingestible device 600 is designed to have anouter housing with a first end 602A, a second end 602B, and a wall 604extending longitudinally from the first end 602A to the second end 602B.The ingestible device 600 has an opening 606 in the housing, whichallows samples to enter the ingestible device 600 from the surroundingenvironment. The ingestible device 600 has an inlet region 608 connectedto the opening 606. The inlet region 608 is connected to an entry port610 of a sampling chamber 612. The inlet region 608 is divided intothree portions. A first portion 608A of the inlet region 608 isconnected to the opening 606 and a second portion 608B, and a thirdportion 608C is connected to the entry port 610 of the sampling chamber612. The second portion 608B connects the first portion 608A to thethird portion 608C, and may contain a moveable valve 614 that is used toprevent samples from flowing through the inlet region 608, and isolatethe first portion 608A of the inlet region 608 from the third portion608C of the inlet region 608.

The ingestible device 600 has a mechanical actuator 624 coupled to themoveable valve 614. In some embodiments, a microprocessor ormicrocontroller is configured to control the mechanical actuator 624 andmove the moveable valve 614 between an open and a closed position. Forexample, the microcontroller may be configured to move the moveablevalve 614 into an open position after the ingestible device reaches aparticular location within the GI tract. In some embodiments, themechanical actuator may be driven by a set of batteries or other powersource located within the ingestible device 600. When the moveable valve614 is moved into an open position, a sample may be allowed to flowthrough the inlet region 608, and enter the sampling chamber 612 throughthe entry port 610. When the moveable valve 614 is in a closed position,the sample is prevented from flowing through the inlet region 608 andreaching the sampling chamber 612 from the opening 606.

For illustrative purposes, FIG. 6 depicts the moveable valve 614 as adiaphragm valve, which uses a mechanical actuator 624 to move a flexiblediaphragm in order to seal or unseal an aperture in the second portion608B of the inlet region 608, which may effectively block or unblock theinlet region 608. However, it will be understood that, in someembodiments, the moveable valve 614 may be a different type of valve.For example, in some embodiments the moveable valve 614 may be replacedby a pumping mechanism, such as the pumping mechanism described inrelation to FIG. 9. As another example, in some embodiments the moveablevalve 614 is replaced with an osmotic valve, similar to the embodimentsdescribed in relation to FIGS. 3, 4, and 5. Several examples of otherdifferent valve types are described in relation to FIG. 7.

The sampling chamber 612 of the ingestible device 600 has an exit port616 located on the opposite end of the sampling chamber 612 from theentry port 610. In general, the exit port 616 may be located anywherewithin the sampling chamber 612. The exit port 616 is configured toallow air or gas 618 to exit the sampling chamber 612, while preventingat least a portion of the sample obtained by the ingestible device 600from exiting the sampling chamber 612. For example, the exit port 616may include a gas-permeable membrane, which allows the gas 618 to exitthe sampling chamber 612, but which would prevent a liquid or solidsample from leaving the sampling chamber 612 through the exit port 616.Allowing the gas 618 to exit the sampling chamber 612 may preventpressure from building up within the sampling chamber 612 as the sampleenters through the entry port 610. This may result in the sample beingdrawn into the sampling chamber 612 more easily, and result inincreasing the overall volume of the sample able to be collected by theingestible device 600, and increasing the ease with which the sample isbrought into the sampling chamber 612.

The ingestible device 600 includes a one-way valve 620 as part of theexit port 616. This valve may prevent the gas 618 from re-entering thesampling chamber 612. However, in some embodiments the one-way valve 620may be excluded from the ingestible device 600. In some embodiments, theexit port 616 includes a gas permeable membrane. This gas permeablemembrane may lose its permeability when it is placed in contact with thesample. For example, the gas permeable membrane may include a spongymaterial that allows the gas 618 to exit the sampling chamber 612through the exit port 616. Once the spongy material becomes moistthrough contact with the sample, it may become no longer gas permeable,or the permeability may be greatly reduced, thereby preventing the gas618 from reentering the sampling chamber 612. In some embodiments, thegas permeable membrane may include expanded polytetrafluorethylene,polypropylene, or the like. In some embodiments, the material used tomake the gas permeable membrane may be filter-like, as opposed tosponge-like materials. Generally, the gas permeable membrane may be madeof any material that allow gas to permeate, but which prevents liquidfrom flowing through the membrane due to sufficient resistance orsurface tension effects.

In the ingestible device 600, the exit port 616 is connected to a volumewithin the housing of ingestible device 600 outside of the samplingchamber. Depending on the manufacturing process used to produce theingestible device 600, the volume within the housing of the ingestibledevice 600 may contain air or some other type of gas.

The ingestible device 600 includes an outlet port 622, which isconnected to the volume within housing of the ingestible device 600. Theoutlet port 622 may provide a path for the gas 618 to exit theingestible device 600 and be released into the environment surroundingthe ingestible device 600. This may be advantageous when the volume ofgas 618 is relatively large, since it may prevent pressure from buildingup within the housing of the ingestible device 600. In some embodiments,the ingestible device 600 does not include an outlet port 622, and thegas 618 stays inside the volume of the ingestible device 600. In someembodiments, the outlet port 622 is directly or indirectly connected tothe exit port 616, for example, by a tube or channel. In someembodiments, the exit port 616 leads directly from the sampling chamber612 to an opening in the ingestible device 600, and the exit port 616may effectively replace the outlet port 622. In some embodiments, theoutlet port 622 may contain a gas permeable membrane, a one-way valve, ahydrophobic channel, or some other mechanism to avoid unwanted material,(e.g., fluids and solid particulates from within the GI tract), fromentering the ingestible device 600 through the outlet port 622.

In some embodiments, the ingestible device 600 may include a sensorwithin or proximate to the sampling chamber 612. For example, thissensor may be used to detect various properties of a sample containedwithin the sampling chamber 612, or this sensor may be used to detectthe results of an assay technique applied to the sample contained withinthe sampling chamber 612.

In some embodiments, a hydrophilic sponge is located within the samplingchamber 612, and the hydrophilic sponge may be configured to absorb thesample as the sample enters the sampling chamber 612. In someembodiments, the hydrophilic sponge fills a substantial portion of thesampling chamber 612, and holds the sample for an extended period oftime. This may be particularly advantageous if the sample is collectedfrom the ingestible device 600 after the ingestible device 600 exits thebody. In some embodiments, the hydrophilic sponge is placed on onlycertain surfaces or fills only certain portions of the sampling chamber612. For example, it may be possible to line certain walls (or allwalls) of the sampling chamber 612 with a hydrophilic sponge to assistin drawing in the sample, while leaving some (or none) of the walls ofthe sampling chamber 612 uncovered. Leaving walls uncovered may allowthe use of diagnostics or assay techniques that involve a relativelyun-obscured optical path. An example of such an embodiment is describedin detail in relation to FIG. 8. In some embodiments, the spongematerial may be placed on all walls of the sampling chamber 612. Thismay prevent unwanted ambient light from entering the sampling chamber612, which may be useful for certain types of low light detectionassays. In some embodiments, an opaque material is used to cover some orall sides of the sampling chamber 612. This may also prevent unwantedambient light from entering the sampling chamber 612.

In some embodiments, the ingestible device 600 may include a sealedvacuum chamber connected to the exit port 616, or connected directly orindirectly to the sampling chamber 612. The sealed vacuum chamber mayhave an internal pressure that is substantially lower than ambientpressure of the sampling chamber 612 and/or the inlet region 608. Inthese embodiments, the ingestible device 600 unseals the vacuum chamberin order to reduce the pressure within the sampling chamber. This changein pressure may force the sample to be sucked into the sampling chamber,or allow the sample to be drawn into the sampling chamber quickly.

For simplicity, FIG. 6 depicts only a single sampling chamber 612, butit will be understood that the inlet region 608 may be connected tomultiple sampling chambers arranged throughout the device, each of whichmay be controlled independently through the use of one or more valves.For example, in some embodiments there may be one or more sub-chambersconnected to the inlet region 608. Each of the sub-chambers may beconfigured to hold samples gathered from within the GI tract, and keepthose samples isolated. In general, any type of valve or other suitablemechanism may be used to isolate samples contained in the sub-chambers,including any of the valves or mechanisms described in relation to FIGS.1-5. In some embodiments, the ingestible device 600 distributesdifferent samples into each of the different sub-chambers at differenttimes, or from different locations within the GI tract. For example, theingestible device 600 may accomplish this by opening up a valve toconnect the interior of inlet region 608 to the appropriate sub-chamberbefore opening up the inlet region 608 to draw in the sample from theopening 606 in the housing.

FIG. 7 depicts different types of moveable valves that may beincorporated into an ingestible device, such as the ingestible devices100, 200 or 600. The ingestible device 702 illustrates how a pin valvemay be used as a moveable valve (e.g., as moveable valve 614 ofingestible device 600 (FIG. 6)), with diagram 702A showing the pin valvein a closed position, and diagram 702B showing the pin valve in an openposition. In the ingestible device 702, a mechanical actuator may beconfigured to move the pin valve linearly in order to switch between anopen position and a closed position. For example, in diagram 702A, theingestible device 702 has a pin inserted into the inlet port, therebypreventing the sample from flowing into the sampling chamber from theopening in the ingestible device 702. In diagram 702B, the ingestibledevice 702 has a pin that has been removed from the inlet port, allowingthe sample to flow freely into the sampling chamber from the opening inthe ingestible device 702. In order to generate linear motion, themechanical actuator may be a linear actuator, such as a solenoid.Alternately, the mechanical actuator may be a rotatory actuator, and therotation may be converted into a linear motion. One skilled in the artwill understand that this may be done any number of ways, for example,by coupling the mechanical actuator to a ball screw mechanism, athreaded lead nut and lead screw mechanism, a rack and pinion mechanism,or the like.

Ingestible device 704 illustrates how a rotary valve may be used as amoveable valve (e.g., as moveable valve 614 of ingestible device 600(FIG. 6)), with diagram 704A showing the rotary valve in a closedposition, and diagram 704B showing the rotary valve in an open position.In diagram 704A, the ingestible device 704 has a rotary pin orientedsuch that the sample is prevented from entering the sampling chamberfrom the opening in the ingestible device 704. In diagram 704B, theingestible device 704 has a rotary pin that has been rotated into anorientation where the sample is free to flow into the sampling chamberfrom the opening in the ingestible device 704. In order to operate therotary valve, the mechanical actuator in ingestible device 704 may be arotatory actuator, which is capable of rotating the rotary pin to switchbetween the open position and the closed position.

Ingestible device 706 illustrates how a flexible diaphragm, or diaphragmvalve, may be used as a moveable valve (e.g., as moveable valve 614 ofingestible device 600 (FIG. 6)), with diagram 706A showing the diaphragmvalve in a closed position, and diagram 706B showing the diaphragm valvein an open position. In diagram 706A, the ingestible device 706 has adiaphragm valve in a closed position, with the flexible diaphragm beingpressed against an aperture in the inlet region due to the pressuregenerated by the mechanical actuator against the flexible diaphragm.This may effectively block a sample from flowing through the inletregion, and thereby preventing a sample from entering the samplingchamber from the opening in the ingestible device 706. In diagram 706B,the ingestible device 706 has a diaphragm valve in an open position,with the pressure removed from the flexible diaphragm. The diaphragmreturns to a position away from the aperture in the inlet region,allowing a sample to flow freely into the sampling chamber from theopening the in ingestible device 706.

In some embodiments, ingestible device 706 has a spring mechanism nearthe diaphragm or in direct contact with the diaphragm. The springmechanism may apply pressure to the diaphragm to oppose the pressureapplied by the mechanical actuator, which may cause the flexiblediaphragm to be moved into an open position when the mechanical actuatoris not applying pressure to the flexible diaphragm. Additionally, thismay ensure that the diaphragm valve remains open when the mechanicalactuator is not applying pressure across the flexible diaphragm.

In some embodiments, moving the mechanical actuator from a closedposition to an open position causes a volume of the inlet region withinthe ingestible device to increase. This may cause the pressure withinthe inlet region to be reduced, generating suction to draw a sample intothe inlet region. Similarly, moving the mechanical actuator from an openposition to a closed position may cause the volume of the inlet regionto be reduced. This may cause the pressure within the inlet region to beincreased, pushing the sample out of the inlet region. Depending on thedesign of the inlet region, the mechanical actuator, and the moveablevalve, this may push the sample into the sampling chamber rather thanpushing the sample back through the opening in the ingestible device. Anexample of such a design is described in greater detail in relation toFIG. 9.

FIG. 8 illustrates an example of a sampling mechanism that may beincorporated into an ingestible device, such as the ingestible devices100, 200, 600, and 702-706. The sampling mechanism 800 is partiallylined with hydrophilic sponges 802A and 802B. In between the hydrophilicsponges 802A and 802B is a testing region 804 within the samplingmechanism 800. The hydrophilic sponges 802A and 802B attract a liquid orfluid sample 806, and may draw the sample 806 into the samplingmechanism 800. As the hydrophilic sponges 802A and 802B are saturatedwith the sample 806, a meniscus 808 is formed at the end of the sample806, between the hydrophilic sponges 802A and 802B. This system may beuseful for acquiring particularly viscous samples, which may havedifficulty flowing into the sampling mechanism 800 naturally.

The sampling mechanism 800 includes an exit port 810 connected to achannel 812. As the sample 806 is drawn into the sampling mechanism 800,air or gas contained in the sampling mechanism 800 may be pushed out ofthe sampling mechanism 800 through the exit port 810 and into thechannel 812. This may avoid gas being trapped within the samplingmechanism 800, which in turn may avoid pressure building inside of thesampling mechanism 800 and preventing the sample 806 from being drawninto the testing region 804.

In some embodiments, the sampling mechanism 800 may not include an exitport 810 or a channel 812, and any air or gas in the sampling mechanism800 may be allowed to remain within the sampling mechanism 800. In someembodiments, the sampling mechanism 800 may be filled with a lowpressure vacuum, attached to a pump or other mechanism to create avacuum, or attached to a sealed chamber containing a low pressure vacuumthat may be unsealed. The use of a vacuum may allow the samplingmechanism 800 to forcibly draw in a sample.

In some embodiments, an ingestible device may include sensors ordiagnostics to study the sample 806 contained within the samplingmechanism 800. Because there is no sponge material on the front and backwalls of the testing region 804, information about the sample 806contained within the testing region 804 may be gathered by using sensorsand/or assay techniques that involve a clear optical path, which wouldotherwise be obscured by a sponge (e.g., the hydrophilic sponges 802Aand 802B). For example, light sources and/or optical sensors may beplaced near the front and/or back walls in order to test opticalproperties of the sample, or to detect the results of certain assaytechniques.

It will be understood by those skilled in the art that the samplingmechanism 800 depicted in FIG. 8 is merely illustrative, and the generaltechniques described in relation to FIG. 8 may be applied to a widerange of different chambers, channels, and fluid pathways, andincorporated into a wide range of different ingestible devices.Furthermore, in some embodiments, the overall geometry of FIG. 8 and thepositioning of the sponges and the testing area may be altered. Forexample, the sponge may be formed in the shape of hollow tubes, withtesting areas located in the middle of each tube. In this case, therewould be a clear optical path from one end of the tube to the other.

FIG. 9 illustrates a pumping mechanism 900 that may be incorporated intoan ingestible device, including certain embodiments of ingestibledevices 100, 200, 600, and 702-706. For illustrative purposes, thepumping mechanism 900 may be described in the context of an ingestibledevice similar to ingestible device 600 (FIG. 6). When it isincorporated into an ingestible device similar to ingestible device 600,the pumping mechanism 900 may function as a moveable valve (e.g.,moveable valve 614 of ingestible device 600), and control the ability ofsamples to flow between the opening 606 in the housing and the entryport 610 of the sampling chamber 612. Additionally, the pumping chamber904 of the pumping mechanism 900 may form part of the second portion608B of the inlet region 608. However, the general structure andprinciples of pumping mechanism 900 are not limited to the ingestibledevices described in this disclosure, and they may be applied to a widerange of ingestible devices.

Pumping mechanism 900 is designed to draw in a sample through a firstopening 902 into a pumping chamber 904, and push a portion of the sampleout of the pumping chamber 904 through a second opening 906. In someembodiments, the first opening 902 may be connected directly orindirectly to an opening in the housing of an ingestible device. Forexample, an inlet region (e.g., the first portion 608A of the inletregion 608 of the ingestible device 600 (FIG. 6)) may connect an openingin the housing of an ingestible device (e.g., the opening 606 in thehousing of ingestible device 600 (FIG. 6)) to the first opening 902. Insome embodiments, the second opening 906 is connected directly orindirectly to a sampling chamber of an ingestible device. For example,the second opening 906 may be connected to an entry port of a samplingchamber (e.g., connected via the third portion 608C of the inlet region608 to the entry port 610 of the sampling chamber 612 of the ingestibledevice 600 (FIG. 6)).

The pumping mechanism 900 features a moveable pump head 908 containedwithin the pumping chamber 904. The protrusion 908A of the moveable pumphead 908 is shaped to fit within the first opening 902, or otherwiseblock the first opening 902. The base 908B of the moveable pump head 908is able to cover the second opening 906 or otherwise block the secondopening 906. Moreover, the protrusion 908A and the base 908B of themoveable pump head 908 are sized and oriented from each other in such amanner such that when the protrusion 908A blocks the first opening 902,the base 908B may simultaneously block the second opening 906 or leavethe second opening 906 unblocked. Furthermore, when the base 908B blocksthe second opening 906, the protrusion 908A may always be configured toalso block the first opening 902.

As the moveable pump head 908 is moved up and down, the openings 902 and906 may be sealed or unsealed, switching the pumping mechanism 900across an open position, a partially closed position, and a closedposition. In the open position (as is shown in the diagram 912), boththe first opening 902 and the second opening 906 are unsealed or open.In the partially closed position (as is shown in the diagram 914, themoveable pump head 908 is positioned to only seal the first opening 902,while leaving the second opening 906 open. Finally, in the closedposition (as is shown in the diagrams 910 and 918), both the firstopening 902 and the second opening 906 are sealed.

In some embodiments, the moveable pump head 908 may be connected to amechanical actuator (e.g., the mechanical actuator 624 of the ingestibledevice 600 (FIG. 6)), which may be configured to move the moveable pumphead 908 linearly up and down. For example, the moveable pump head 908may be located on the end of a shaft that is attached to the mechanicalactuator. In some embodiments, the mechanical actuator and thepositioning of the moveable pump head 908 may be controlled by amicrocontroller or microprocessor located within the ingestible device.For example, a microcontroller may be configured to move the pump head908 and begin pumping a sample through the pumping chamber 904 onlyafter the ingestible device has reached a particular location within theGI tract.

Diagram 910 depicts the pumping mechanism 900 in a fully closedposition. When the pumping mechanism 900 is in the fully closedposition, the protrusion 908A of the moveable pump head 908 may bepositioned within the first opening 902, and the base 908B of themoveable pump head 908 may be positioned adjacent to the second opening906. In the fully closed position, the positioning of the moveable pumphead 908 may effectively prevent a sample from entering or exiting thepumping chamber 904 from the openings 902 or 906.

Diagram 912 depicts the pumping mechanism 900 in an open position. Whenthe pumping mechanism 900 is in the open position, the moveable pumphead 908 is moved away from the first opening 902, moving the protrusion908A of the moveable pump head 908 out of the first opening 902, andmoving the base 908B of the moveable pump away from the second opening906. In this position, the pumping mechanism 900 may allow one or moresamples to enter the pumping chamber 904 through the first opening 902,and exit the pumping chamber 904 through the second opening 906. Becausethe effective volume of the pumping chamber 904 increases when themoveable pump head 908 is moved away from the first opening 902, thepumping mechanism 900 may draw a sample into the sampling chamberthrough the first opening 902 when transitioning from a closed positiondepicted in the diagram 910 to an open position depicted in the diagram912. In some embodiments, a one-way valve may be incorporated into aningestible device to prevent samples from being drawn into the pumpingchamber 904 through the second opening 906 when the pumping mechanism900 transitions between the closed position and the open position. Thismay ensure that the only sample entering the pumping chamber 904 isdrawn in through the first opening 902.

Diagram 914 depicts the pumping mechanism 900 in a partially closedposition. When the pumping mechanism 900 is in the partially closedposition, the protrusion 908A of the moveable pump head 908 ispositioned adjacent to the first opening 902, or just inside the firstopening 902. In this position, the protrusion 908A of the moveable pumphead 908 effectively seals off the first opening 902, preventing any ofthe sample remaining in the pumping chamber 904 from exiting pumpingchamber 904 via the first opening 902. In this position, the base 908Bof the moveable pump head 908 is positioned away from the second opening906. This may allow any sample remaining in the pumping chamber 904 toexit the pumping chamber 904 through the second opening 906. Forexample, if the second opening 906 is connected to an entry port of asampling chamber (e.g., connected via the third portion 608C of theinlet region 608 to the entry port 610 of the sampling chamber 612 ofthe ingestible device 600 (FIG. 6)), this may allow the sample to flowfreely from the pumping mechanism 900 into the sampling chamber via theentry port.

Diagram 916 depicts the pumping mechanism 900 as it transitions betweenthe partially closed position to the fully closed position. As thepumping mechanism 900 moves into the fully closed position, the moveablepump head 908 forces any of remaining sample contained within thepumping chamber 904 out of the pumping chamber 904 through the secondopening 906. As this happens, the protrusion 908A of the moveable pumphead 908 remains within the first opening 902, blocking it off andpreventing the sample from exiting the pumping chamber 904 through firstopening 902. By comparison, the base 908B of the moveable pump head 908does not fully cover the second opening 906, and the sample is free toexit the pumping chamber 904 through the second opening 906. Incombination, this may result in a majority of the sample remaining inthe sampling chamber being forced through the second opening 906 as thepumping mechanism 900 moves from the partially closed position depictedin diagram 914 to the fully closed position depicted in diagram 918.

Diagram 918 depicts the pumping mechanism 900 in the fully closedposition, similar to diagram 910. As noted before, in the fully closedposition the moveable pump head 908 is positioned to seal off theopenings 902 and 906, which may prevent a sample from entering orexiting the pumping chamber 904 from the openings 902 or 904. Ingeneral, the pumping mechanism 900 may cycle between the closed positiondepicted in diagrams 910 and 918 and the open position depicted indiagram 912 any number of times in order to draw additional samples intothe pumping chamber 904 through the first opening 902, and force thesamples out of the pumping chamber 904 through the second opening 906.

Although FIG. 9 depicts the protrusion 908A of the moveable pump head908 located in the center of the moveable pump head 908, the location ofthe protrusion 908A may be anywhere on the moveable pump head 908. Forexample, the protrusion 908A of the moveable pump head 908 and the firstopening 902 may be positioned on the side of the pumping chamber 904. Insome embodiments, the moveable pump head 908 is split into two pieces,which may be controlled by one or more actuators. For example, theprotrusion 908A and the base 908B may be two separate pieces, each ofwhich is moved using a different actuator. This may allow the firstopening 902 to be sealed and unsealed independently from the volume ofthe pumping mechanism 900 being increased or decreased.

For illustrative purposes, the diagrams 910-918 depict the base 908B ofthe moveable pump head 908 being used to cover or otherwise block thesecond opening 906. However, in some embodiments, the moveable pump head908 may not cover, fit within, or otherwise block the second opening906, and it will be understood by one skilled in the art that the secondopening 906 does not need to be partially or fully blocked in order topush a sample through the second opening 906. For example, the moveablepump head 908 may not include a base 908B at all. Instead, the moveablepump head 908 may be made of a flexible material that forms a seal withthe underside of the pumping chamber 904. In this case, the moveablepump head 908 may be moved up and down in a manner similar to a plungerin order to change the effective volume of the pumping chamber 904. Whenthe volume decreases, the sample is at least partially forced out of thepumping chamber 904 through the second opening 906.

In general, incorporating the pumping mechanism 900 into an ingestibledevice may not impair the function of the openings, ports, valves,membranes, sampling chambers, or other structures of the ingestibledevice, and any of the teachings or embodiments described in conjunctionwith the ingestible devices 100, 200, 600, or 702-706 may be combined indifferent embodiments of an ingestible device along with the pumpingmechanism 900. For example, the pumping mechanism 900 may replace thefirst valve 214 in the ingestible device 200 (FIG. 2), and may be usedto force the sample into the sampling chamber 212. As an alternateexample, the pumping mechanism 900 may be used to force samples into thesampling chamber 304 of the osmotic valve mechanism 300 (FIG. 3). Asanother example, the pumping mechanism 900 may be incorporated into anembodiment of the ingestible device 600 (FIG. 6) where the exit port 616is not included, and the pumping mechanism 900 may be used to force thesample into the sampling chamber 612 despite the pressure that mayresult from air or gas 618 being trapped within the sampling chamber612.

FIG. 10 illustrates, in a highly schematic fashion, an ingestible device1000 having a housing 1010 that includes a first end 1012 and a secondend 1014 opposite first end 1012. Housing 1010 also includes a wall 1016that connects first end 1012 and second end 1014. Wall 1016 has anopening 1018 that allows fluid from an exterior of the ingestible device1000 (e.g., from the GI tract) and into an interior of ingestible device1000.

FIG. 11 depicts a cross-sectional view of a portion of the interior ofingestible device 1000. As shown in FIG. 11, the interior of ingestibledevice 1000 includes a valve system 1100 and a sampling system 1200.Valve system 1100 is depicted as having a portion that is flush with theopening 1018 so that valve system 1100 prevents fluid exterior toingestible device 1000 from entering sampling system 1200. However, asdescribed in more detail below with reference to FIGS. 12-16, valvesystem 1100 can change position so that valve system 1100 allows fluidexterior to ingestible device 1000 to enter sampling system 1200.

FIGS. 12 and 16 illustrate valve system 1100 in more detail. As shown inFIG. 12, valve system 1100 includes an actuation mechanism 1110, atrigger 1120, and a gate 1130. In FIGS. 12 and 16, a leg 1132 of gate1130 is flush against, and parallel with, housing wall 1016 so that gateleg 1132 covers opening 1018 to prevent fluid exterior to ingestibledevice 1000 (e.g., fluid in the GI tract) from entering the interior ofingestible device 1000. A protrusion 1134 of gate 1130 engages a lip1122 of trigger 1120. A peg 1124 of trigger 1120 engages a wax pot 1112of actuation mechanism 1110. Referring to FIG. 16, a biasing mechanism1140 includes a compression spring 1142 that applies an upward force ongate 1130. Biasing mechanism 1140 also includes a torsion spring 1144that applies a force on trigger 1120 in the counter-clockwise direction.In FIGS. 12 and 16, the force applied by torsion spring 1144 iscounter-acted by the solid wax in pot 1112, and the force applied bycompression spring 1142 is counter-acted by lip 1122.

FIG. 13A and FIG. 13B show an embodiment of the manner in whichactuation mechanism 1110 actuates movement of trigger 1120. Similar toFIGS. 12 and 16, FIG. 13A shows a configuration in which peg 1124applies a force against solid wax pot 1112 due to torsion spring 1144,and in which the solid nature of wax pot 1112 resists the force appliedby peg 1124. A control unit 1150 is in signal communication with valvesystem 1100. During use of ingestible device 1000, a control unit 1150receives a signal, indicating that the position of valve system 1100should change, e.g., so that ingestible device 1000 can take a sample ofa fluid in the GI tract. Control unit 1150 sends a signal that causes aheating system 1114 of actuation system 1100 to heat the wax in pot 1112so that the wax melts. As shown in FIG. 13B, the melted wax is not ableto resist the force applied by peg 1124 so that, under the force oftorsion spring 1144, trigger 1120 moves in a counter-clockwise fashion.

FIGS. 14A and 14B illustrate the interaction of trigger 1120 and gate1130 before and after actuation. As shown in FIG. 14A, when wax pot 1302is solid (corresponding to the configuration shown in FIG. 13A),protrusion 1134 engages lip 1122, which prevents the force ofcompression spring 1142 from moving gate 1130 upward. As shown in FIG.14B, when the wax in pot 1112 melts (FIG. 13B), trigger 1120 movescounter-clockwise, and lip 1122 disengages from protrusion 1134. Thisallows the force of compression spring 1142 to move gate 1130 upward. Asseen by comparing FIG. 14A to FIG. 14B, the upward movement of gate 1130results in an upward movement of an opening 1136 in gate leg 1132.

FIGS. 15A and 15B illustrate the impact of the upward movement ofopening 1136 on the ability of ingestible device 1000 to obtain asample. As shown in FIG. 15A, when the wax in pot 1112 is solid (FIGS.13A and 14A), opening 1136 in is not aligned with opening 1018 in wall1016 of ingestible device 1000. Instead, gate leg 1132 covers opening1018 and blocks fluid from entering the interior of ingestible device1000. As shown in FIG. 15B, when the wax in pot 1112 is melted andtrigger 1120 and gate 1130 have moved (FIGS. 13B and 14B), opening 1136in gate 1130 is aligned with opening 1018 in wall 1016. In thisconfiguration, fluid that is exterior to ingestible device 1000 (e.g.,in the GI tract) can enter the interior of ingestible device 1000 viaopenings 1018 and 1036.

While the foregoing description is made with regard to a valve systemhaving one open position and one closed position (e.g., a two-stagevalve system), the disclosure is not limited in this sense. Rather, theconcepts described above with regard to a two stage valve system can beimplemented with a valve system have more than two stages (e.g., threestages, four stages, five stages, etc.). For example, FIGS. 17A-19Cillustrate cross-sectional views of a three-stage valve system 1700.FIGS. 17A, 18A and 19A illustrate different views of components of valvesystem 1700 in the same position. FIGS. 17B, 18B and 19B illustratedifferent views of components of valve system 1700 in the same position.FIGS. 17C, 18C and 19C illustrate different views of components of valvesystem 1700 in the same position.

As shown in FIGS. 17A-19C, valve system 1700 includes an actuationsystem 1710, a trigger 1720, a gate 1730 and a biasing system 1740.Actuation system 1710 includes a first wax pot 1712, a second wax pot1714, a first heating system 1716 and a second heating system 1718.Trigger 1720 includes a first lip 1722, a second lip 1724, a first peg1726 and a second peg 1728. Gate 1730 includes a gate leg 1732 and aprotrusion 1734. Gate leg 1732 has an opening 1736. Biasing system 1740includes a compression spring 1742 and a torsion spring 1744. Inaddition, the ingestible device includes a control unit 1750.

As shown in FIGS. 17A, 18A and 19A, in the first stage, protrusion 1734engages first lip 1722, and first peg 1726 engages first wax pot 1712.Compression spring 1742 applies an upward force on gate 1730, andtorsion spring 1744 applies a force on trigger 1720 in thecounter-clockwise direction. The force applied by torsion spring 1744 iscounter-acted by the solid wax in first pot 1712, and the force appliedby compression spring 1742 is counter-acted by first lip 1722. Opening1736 is not aligned with opening 1018.

FIGS. 17B, 18B and 19B illustrate the configuration in a second stage,after control unit 1750 sends a signal to first heating system 1716 tomelt the wax in first pot 1712. In the second stage, trigger 1720 hasmoved counter-clockwise relative to its position in the first stage.First peg 1726 is positioned in first pot 1712 because the melted waxcannot prevent this movement. Further counter-clockwise movement oftrigger 1720 is prevented by the engagement of second peg 1728 with thesolid wax in second pot 1714. With the counter-clockwise movement oftrigger 1720, first lip 1722 disengages from protrusion 1734, and gate1730 moves upward so that opening 1736 in leg 1732 is aligned withopening 1018. Further upward movement of gate 1730 is prevented by theengagement of protrusion 1734 with second lip 1724.

FIGS. 17C, 18C and 19C illustrate the configuration in a third stage,after control unit 1750 sends a signal to second heating system 1718 tomelt the wax in second pot 1714. In the third stage, trigger 1720 hasmoved counter-clockwise relative to its position in the second stage.Second peg 1728 is positioned in second pot 1714 because the melted waxcannot prevent this movement. Further counter-clockwise rotation isprevented by the engagement of first and second pegs 1726 and 1728,respectively with first and second pots 1712 and 1714, respectively.Protrusion 1734 is disengaged from second lip 1724, allowing the forceof compression spring 1742 to move gate 1730 upward so that opening 1736is no longer aligned with opening 1018.

FIG. 20 illustrates another embodiment of a three stage valve system2000 that can be used in an ingestible device. Valve system 2000 that issimilar to valve system 1700 except that actuation system 2010 includesthree includes wax pots 2012, 2014 and 2016, respectively, that define atriangle, and trigger 2020 includes three pegs 2022, 2024 and 2026,respectively, that define a corresponding triangle. Actuation system2010 is controlled using a control unit 2050. Actuation system 2010 alsoincludes a first heating system 2018 that heats the wax in pots 2012 and2014 and so that pegs 2022 and 2024 enters their corresponding pot,causing valve system 2000 to move from its first stage to its secondstage. Actuation system 2010 also includes a second heating system 2028that heats the wax in pot 2016 so that pegs 2026 enters pot 2016,causing valve system 2000 to move from its second stage to its thirdstage.

In the foregoing discussion, embodiments actuating systems are describedthat include one or more wax pots and corresponding heating systems. Butthe disclosure is not limited to such actuating systems. Generally, anyactuating system can be used that will provide an appropriate force toresist counter-clockwise movement of the trigger when desired and toremove that force when desired. Examples of such actuation systemsinclude a pot with a silicon or wax seal. A control unit may be used torupture the seal and allow counter clock-wise movement of the trigger.Additionally, or alternatively, the actuation mechanism may usedissolvable coating to that dissolves over time or in the presence of asubstance. As the coating dissolves, the trigger may move further in thecounter clock-wise direction. Other actuation mechanisms may also applyan attractive force rather than remove a resistive force. For example,the actuation mechanism may include magnetic pegs and slidable magnetsThe magnets may be located behind the pots or may slide to a positionbehind the pots when the valve system should change stages. As themagnets behind the pots slide into range of the magnetic trigger pegs,the trigger moves in the counterclockwise direction due to theattractive force between the magnetic peg and the magnets. The slidingmechanism to move the slidable magnets may be powered by an osmoticpump, a pressurized chamber, or any other applicable method of movementpreviously described in other embodiments.

In the discussion above, embodiments of triggers are disclosed thatinclude one or more lips and one or more pegs. However, the disclosureis not limited to such triggers. In general, for example, any triggerdesign can be used that is capable of providing the step-wise movementof the trigger. Such trigger designs include, for example, a releasablelatch coupling or a saw toothed engagement wall. A different embodimentmay utilize a ball in socket joint to engage the trigger and gate, inwhich the “socket” is located on the trigger. It is to be noted thatsuch designs need not be based on counter-clockwise movement and may be,for example, designed for the controlled movement of the trigger in oneor more of various degrees of freedom. For example, rather than rotate,the trigger may be configured to slide laterally to push a peg of thetrigger into a melted wax pot.

The discussion above describes embodiments of gates that include aprotrusion and a leg with an opening. The disclosure is not limited tosuch designs. Generally, any appropriate arrangement can be used so longas it provides the desired step-wise controlled movement of an openingto the interior of the ingestible device. Exemplary designs include agate that is capable of responding to or applying magnetic forces on thetrigger. A saw toothed pattern may also provide a step-wise gatemovement. Additionally, embodiments include a latch designed toreleasably couple the gate to the trigger. A different embodiment mayutilize a ball in socket joint in which the “ball” is located on thegate. Optionally, a gate can include one or regions that include one ormore appropriate sealing materials positioned to cover the opening inthe housing of the ingestible device when the gate is positioned toprevent fluid exterior to the ingestible device from entering theinterior of the device via the opening in the housing of the ingestibledevice.

In the foregoing discussion, embodiments of biasing systems aredescribed that include a compression spring and a biasing spring.However, the disclosure is not limited in this sense. In general, anybiasing elements can be used to provide the counter-clockwise force tothe trigger and/or to provide the upward force to the gate. Exemplarybiasing elements include elastic bands, wherein a stretched elastic bandacts similar to a stretched compression spring as described. Additionalbasing mechanisms may include magnets and/or magnetic forces to inducetrigger or gate movement. For example, a magnet may be located above thegate, where, like the constant force of the stretched compressionspring, the magnet also applies a constant attractive force on the gate.

As noted above in addition to a valve system, an ingestible deviceincludes a sampling system. FIGS. 21A and 21B illustrate a partial crosssectional view of ingestible device 1000 with sampling system 1200 andcertain components of valve system 1100. Sampling system 1200 includes aseries of sponges configured to absorb fluid from an opening, move thefluid to a location within the housing, and prepare the fluid fortesting. Preparation for testing may include filtering the fluid andcombining the fluid with a chemical assay. The assay may be configuredto dye cells in the filtered sample. The series of sponges includes awicking sponge 1210, a transfer sponge 1220, a volume sponge 1230, andan assay sponge 1240.

Wicking sponge 1210 absorbs the fluid form the opening in the housingwhen the valve is open i.e. when the inlet and the housing are aligned.The wicking sponge transfers the fluid from the opening to a filter.Wicking sponge 1210 includes a wicking tongue 1212 extended towards thehousing 1016. As shown in FIG. 21A, before actuation of the actuationsystem (FIGS. 13A, 14A, 15A), wicking tongue 1212 is not adjacentopening 1018 in wall 1016 of ingestible device 1000 so that wickingtongue 1212 does not absorb fluid exterior to ingestible device 1000.However, as shown in FIG. 21B, after actuation of the actuation system(FIGS. 13B, 14B, 15B), wicking tongue 1212 is adjacent opening 1018 sothat wicking sponge 1212 absorbs fluid that passes through opening 1018,e.g., fluid from the GI tract. Fluid absorbed by wicking tongue 1212 cantravel through wicking sponge 1210 to a distal end 1214 of wickingsponge 1210. The wicking sponge 1210 and wicking tongue 1212 may be madeof a VF2 sponge, an Ahlstrom M13 sponge, MF/F material, a Carwild IvalonPolyvinyl Alcohol material, or another suitable absorptive material.Optionally, the dimensions of the sponge material may be selected toenable all its desired functions while remaining precisely packagedwithin the capsule. In some embodiments, Carwild Ivalon PolyvinylAlcohol material is cute to the dimensions 1.4 millimeters (height)×6millimeters (width)×8.5 millimeters (length). In certain embodiments,one or more of the following parameters can be considered when selectingan appropriate material and/or its dimension: ability to load one morepreservative materials; desired preservative material(s) to be loaded;capacity to hold one or more dried preservatives; ability to facilitatehydration of one or more dried preservative materials upon contact withone or more GI fluids; capacity to capture fluid (e.g., GI fluid); andswelling properties upon fluid uptake (generally, it is desirable tohave little or no swelling upon fluid uptake).

A cell filter 1250 is located between distal end 1214 of wicking sponge1210 and a first end 1222 of transfer sponge 1220. The cell filter 1250is configured to prevent undesired cells, such as Hela cells, fromentering one or more downstream sponges in sampling system 1200,particularly sponges used in testing. Excluding such undesired cellsenhances the accuracy of various analytical results.

Fluid that passes from wicking sponge 1210 and through cell filter 1250can enter transfer sponge 1220 via its first end first end 1222.Transfer sponge 1220 is configured to move the filtered fluid from cellfilter 1250 to volume sponge 1230 and/or assay sponge 1240.

To allow transfer sponge 1220 to absorb a relatively large volume offluid, transfer sponge 1220 is shaped (e.g., arc-shaped) to provide arelatively long distance between first end 1222 of transfer sponge 1220and a second end 1224 of transfer sponge 1220. Second end 1224 contactsboth volume sponge 1230 and assay sponge 1240 while preventing volumesponge 1230 and assay sponge 1240 from directly contacting each other. Abarrier 1260 is located between first end 1222 and volume sponge 1230 toensure that fluid absorbed in transfer sponge 1220 at first end 1222travels to second end 1224 before being absorbed by volume sponge 1230.Although depicted as being arc-shaped, transfer sponge 1220 can have oneor more different configurations, such as, for example, an extendedstraight line or multiple curves, depending, for example, on the desiredvolume of sample and/or desired transfer speed. In general, the shorterand/or thinner the path of transfer sponge 1220, the quicker thetransfer speed from first end 1222 to second end 1224. The transfersponge 1220 may be made of a VF2 sponge, an Ahlstrom M13 sponge, MF/Fmaterial, or another suitable absorptive material.

Volume sponge 1230 absorbs additional fluid for testing and is in fluidcommunication with assay sponge 1240 via second end 1224 of transfersponge 1220. Volume sponge 1230 can be particularly useful whenfluorescent or optical testing is used. In some embodiments, assaysponge 1240 and transfer sponge 1224 may not individually contain asufficient volume of the sample to attain a confident test result. Thevolume of volume sponge 1230, assay sponge 1240, and second end 1224 ofthe transfer sponge 1220 sum to a sufficient testing volume for optical,and other, tests. Assay sponge 1240 contains a chemical assay that isused to test the sample or to prepare the sample for a test. Once assaysponge 1240 is saturated, the assay chemicals are free to flow fromassay sponge 1240 and interact with sample absorbed by transfer sponge1220 and volume sponge 1230. Volume sponge 1230 and the assay sponge1240 may be made of a VF2 sponge, an Ahlstrom M13 sponge, MF/F material,or another suitable absorptive material. Preferably, the wicking sponge,wicking tongue, transfer sponge, and assay sponge are Ahlstrom M13sponges, and the volume sponge is a VF2 sponge.

Cell filter 1250 can be made from any appropriate material and have anyappropriate dimensions. Exemplary materials include polycarbonate(PCTE), polyethersulfone (PES), polyester (PETE) andpolytetrafluoroethylene (PTFE). In some embodiments, the dimensions ofcell filter 1250 can be about 9.5 millimeters by about 6.5 millimetersby about 0.05 millimeter.

Sampling system 1200 also includes a membrane 1270 located between assaysponge 1240 and a vent 1280 for gases to leave sampling system 1200.Membrane 1270 is configured to allow one or more gases to leave samplingsystem 1200 via an opening 1280, while maintaining liquid in samplingsystem 1200.

FIG. 22 illustrates an embodiment of ingestible device 1000 with arelatively detailed view of both valve system 1100 and sampling system1200. FIG. 22 shows valve system 1100 positioned prior to actuation ofactuation system 1110 (e.g., when configured as shown in FIGS. 13A, 14A,15A and 20A).

FIG. 23 illustrates an embodiment of an ingestible device includingsampling system 1200 and three-stage valve system 1700 positioned in itsthird stage.

FIG. 24 illustrates an embodiment of an ingestible device 1000 includingsampling system 1200 and valve system 2000 positioned in its thirdstage.

FIG. 25 is a highly schematic illustration of an ingestible device 3000that contains multiple different systems that cooperate for obtaining asample and analyzing a sample, e.g., within the GI tract of a subject.Ingestible device 3000 includes a power system 3100 (e.g., one or morebatteries), configured to power an electronics system 3200 (e.g.,including a control system, optionally in signal communication with anexternal base station), and an analytic system 3500.

Exemplary analytical systems include assay systems, such as, forexample, optical systems containing one or more sources of radiationand/or one more detectors. Such systems may use, for example, a lightsource that illuminates and a sample and a detector configured to detectlight that is emitted by the sample (e.g., fluorescence spectroscopy),optical density (e.g., the portion of light that passes through thesample), and/or light that is diffracted by sample (e.g., diffractionoptics). An analytical system may use, for example, ELISA (enzyme-linkedimmunosorbent assay). An analytical system may use, for example, LOCI(luminescent oxygen channeling). An analytical technique may involveincubating and/or diluting a sample before or during theanalysis/assaying of the sample. An analytical technique may involve theuse of staining/dyeing a live cell.

Ingestible device 3000 also includes a sampling system 3400 for takingin a sample from the environment exterior to ingestible device 3000, anda valve system 3300 that regulates the ability of a fluid to accesssampling system 3400.

FIG. 26 provides an exploded view of the ingestible device 3000. FIG. 26includes an exploded view of ingestible device 3000, showing a generalconfiguration of the systems in FIG. 25. FIG. 26 includes power system3100 (e.g., a stack of batteries), electronic system 3200 (e.g., a PCBand associated wiring), valve system 3300, sampling system 3400, andanalytic system 3500.

FIG. 27 illustrates a portion of an ingestible device 4000 with a port4154 b in an open position to the exterior of the ingestible device4000. The ingestible device 400 may include a cylinder-shaped rotatableelement 4150 that includes sampling ports 4154 a-b on the wall of therotatable element 4150. The sampling chamber 4150 is wrapped by a shellelement 4140 with dividers to form a series of dilution chambers 4151a-n between the shell element 4140 and the rotatable element 4150. Inoperation, when the ingestible device 4000 determines the device itselfarrives at a target location within the GI tract, the rotatable element4150 may be rotated into an open position such that an aperture of theshell element 4140 is aligned with the port 4154 b on the wall of therotatable element 4150 and the port 4154 b is exposed to the exterior ofthe ingestible device 4000 through the aperture. In this way, fluid fromthe GI tract can enter the port 4154 b and occupy the volume defined bythe port 154 b. In the embodiment shown in FIG. 24, the port 4154 b maybe a depression on the surface of a rotatable element 4150 and a numberof dilution chambers 4151 a-n are positioned circumferentially aroundthe axis of rotation of the rotatable element 4150. As previouslydiscussed, each of the dilution chambers 4151 a-n may store a dilutionfluid. In one embodiment, the depression is a cylindrical depression.Optionally, the depression may be a rectangular depression, or anyconcave depression forming a regular or irregular shape. In anotherembodiment, the port 4154 b may be connected to a chamber (not shown)within the rotatable element 4150 to create an enlarged space to storethe GI fluid sample from the external environment of the ingestibledevice.

In some embodiments, the ingestible device 4000 may further include acontroller and an actuator. The controller may determine that theingestible device 100 is located at a target location of the GI tract,and then the actuator may trigger the rotation of the rotatable element4150 to align the port 4154 b at the open position to initiate thesampling. For example, the housing of ingestible device 4000 may have apH-sensitive enteric coating to detect or otherwise be sensitive to a pHlevel of the environment external to the ingestible device 4000, basedon which the controller may determine whether the ingestible device hasarrived at a target location. For another example, the ingestible device4000 may include an optical sensing unit that transmits an illuminationto the environment and collects a reflectance, based on which, theregio-specific location of the ingestible device 4000 may be identifiedbased on optical characteristics of the reflectance.

FIG. 28 shows one embodiment of a portion of an ingestible device with aport 4154 b at a first position aligned with a first dilution chamber4151 a. In operation, the rotatable element 4150 may be rotated to alignthe sampling port 4154 b and the first dilution chamber 4151 a such thatthe fluid sample from the GI tract stored within the volume of thesampling port 4154 b can be combined with dilution fluid in the firstdilution chamber to form a first dilution. The first dilution may thenoccupy the combined volume of the port 4154 b and first dilution chamber4151 a. Optionally, the rotatable element 4150 may be subsequentlyrotated to a second position such that the port 4154 b containing aportion of the first dilution is then moved to be aligned and in fluidcommunication with another dilution chamber, e.g., a second dilutionchamber that is next to the first dilution chamber along the rotationaldirection. In this way, the first dilution stored within the port 4154 bmay then again be diluted with the dilution fluid stored within thesecond dilution chamber. Similarly, if the rotatable element 4150 keepsrotating and allows the port 4154 b to be serially aligned with eachdilution chamber, then the original GI fluid sample may be dilutedserially and each dilution chambers 4151 a-n may be left with a dilutedGI fluid sample at a different dilution ratio.

FIG. 29 shows an embodiment of an element 4140 forming part of a set offive dilution chambers (e.g., including 4151 a-b) for surrounding arotatable element (e.g., 4150 in FIGS. 21-22) in an ingestible device asdescribed herein. In one embodiment, the device may contain a singledilution chamber. Alternatively, the device may contain 2, 3, 4, 5, 6,7, 8 or greater than 8 dilution chambers.

In some embodiments, each dilution chamber 4151 a-n may be filled with adilution fluid prior to the ingestible device 4000 being administered.In another embodiment, the dilution fluid may be stored in a separatereservoir (not shown) within the ingestible device 4000. At the timewhen the ingestible device 4000 is determined to be at a target locationwithin the GI tract, a pumping mechanism may pump the dilution fluidinto one or more dilution chambers 4151 a-b via one or more outlet (notshown) of the reservoir.

In some embodiments, the shell element 4140 may have valves or pumps(not shown) between the dilution chambers 4151 a-n. For example, thediluted fluid from a first dilution chamber may be pumped into a seconddilution chamber via a valve between the two chambers.

Devices of the type depicted in FIGS. 27-29 optionally can include asampling system as disclosed herein.

In certain embodiments, an ingestible device includes a microscopicevaluation system. In some embodiments, bacterial cells in a sample maybe first labeled with fluorescent dyes (such as those described herein),and the fluorescently-labeled cells may be imaged and counted by themicroscopic evaluation using an ingestible device as described herein.In other embodiments, the fluorescently-labeled cells are counted asthey pass through an onboard flow system (e.g., microfluidic single cellchanneling). Examples of flow cytometry systems include hydrodynamicfocusing, small diameter capillary tube flow, and rectangular capillarytube flow. As described herein, live bacteria cells are labeled, and theprinciples of flow cytometry are used to quantify labeled cells.Generally speaking, the photons from an incident laser beam are absorbedby the fluorophore and raised to a higher, unstable energy level. Withinless than a nanosecond, the fluorophore re-emits the light at a longerrepresentative wavelength where it is passed through a series ofdichroic filters. This reemitted light can be collected and interpretedas proportional to the number of labeled bacteria cells. In someembodiments, a sheath fluid is not used as part of the flow system tohelp accommodate the volume restrictions of the device. In someembodiments, a rectangular capillary tube is used to achieve asufficiently large cross-sectional area and relatively thin inspectionarea. The flow cytometry optical system operates parallel to thefluidics system and serves to observe the redirection of light passingthrough the cell and delivers information about the bacterial cells. Insome embodiments, rather than using a conventional laser and sphericallenses to focus the light to a point, an LED and cylindrical lenses areused to focus the light to a line across a rectangular capillary tube.In other embodiments, collimating lenses are used to make the lightsource parallel, while cylindrical lenses are used to refine theinspection area. An exemplary optical configuration for this arrangementcan be seen in FIG. 30. In some embodiments, optical filters can beadded to permit the use of fluorophores. The characteristic wavelengthof reemitted light from the fluorophores can be isolated and detectedwith the use of dichroic, bandpass, and short or long wave pass filters.Generally, multiple dichroic lenses and photomultipliers are used,however, due to space limitations, only a single side-scatter detectorand forward scatter detector may be used in certain embodiments.

One of the design challenges of integrating flow cytometry into thedevice is to provide a pumping mechanism. Without moving fluid,individual bacteria cells cannot be identified and accounted for by flowcytometry within a fixed volume of fluid. In some embodiments, a gearmotor is to move fluid through the device. For example, a micromotorcomprising a planetary gearhead (e.g., with a 25:1 reduction) canprovide the desired amount of torque to create fluid flow. In anotherembodiment, a series of piezoelectric resistors embedded in the surfaceof a microfabricated plate is used to create flow. In yet anotherembodiment, a micropump that includes a pair of one-way valves and usesa magnetic pump membrane actuated by an external magnetic field is usedto create flow.

In some embodiments, the system architecture comprises an opening andsealing mechanism combined with a rotary wiper which creates a pressuredriven flow via a gear motor. The gear motor can be used for otherfunctions in the device. As shown in FIG. 31, the components of theoptics and flow chamber systems fit within the device. In someembodiments, the sample fluid is absorbed via a flexible membrane at thetop of the capsule. In some embodiments, the gear motor has 270° ofpermissible travel which serves to open and fill the fluid chamber.During closure, the motor closes the ingress port while simultaneouslypushing the fluid through the rectangular capillary tube where theoptical system is located. The threaded component allows the flexiblemembrane to close and seal the ingress channel without changing thewiper height. In some embodiments, the volume of the sample chamber is25 μL, 50 μL, 75 μL or more. In some embodiments, two or more samplesare taken from the GI tract to procure a sufficient sample size.Referring to FIG. 31, an LED on the left side of the capillary tube andthe two low-light detectors on the right for capturing forward and sidescatter are shown. Once the fluid passes through the capillary tube, itexits the capsule via a one-way valve. In certain embodiments, the flowsystem allows for the detection of cell size and internal cellcomplexity, in addition to cell quantitation.

The foregoing discussion is not exhaustive with respect to variousingestible device designs, either with respect to sampling componentryor absorbent (sponge) design.

As an example, while ingestible devices have been described that includeone or more optical systems incorporated into the ingestible device, insome embodiments, an ingestible device does not include an opticalsystem. Optionally, such ingestible devices may also not include anyother analytical componentry. In embodiments of an ingestible devicewhich do not include an optical system and/or other analyticalcomponentry, there may be more room inside the ingestible device tostore one or more samples.

Exemplary ingestible devices are provided in U.S. Ser. No. 14/460,893,which is incorporated by reference herein.

FIG. 32 shows a partial view of an exemplary embodiment of an ingestibledevice 5010 in which a portion of the enclosure of ingestible device5010 has been removed. Ingestible device 5010 may be used for collectingsubstances. Ingestible device 5010 may generally be in the shape of acapsule, like a conventional pill. Accordingly, the shape of ingestibledevice 5010 provides for easier ingestion and is also familiar tohealthcare practitioners and patients.

The structure of ingestible device 5010 includes first portions andsecond portions 5012 and 5014. First portion 5012 includes controlelectronics, a power supply, and a communication system. Second portion5014 is generally configured to interact with the GI tract, such as, forexample but not limited to, sample collection, substance delivery andenvironmental monitoring. Second portion 5014 includes a storagesub-unit 16 with one or more chambers 5018 and a chamber enclosure 5020that encloses or overlays a storage sub-unit 5016. Each chamber 5018 hasa corresponding chamber opening 5022. Chamber enclosure 5020 has anaccess port 5024. In this example embodiment, ingestible device 5010includes three chambers 5018, but there can be other embodiments thathave one, two or more than three chambers 5018.

FIGS. 33A-33C illustrate operation of ingestible device 5010. Generally,chamber enclosure 5020 operates as a “closed-loop” revolver mechanism.Chamber enclosure 5020 rotates, in a controlled manner, to align theaccess port 5024 with each of chamber openings 5022 for collecting, attargeted locations, samples of the contents in the GI into correspondingchamber 5018, and/or for delivering substances stored in chambers 5018to targeted locations within the body.

Generally, during collection of samples, the rotation of chamberenclosure 5020 may be described as a “closed-loop” revolver mechanismbecause each chamber opening 5022 is exposed only once during thepassage of ingestible device 5010 within the body in order to avoidcross-contamination of the collected samples. In other words, in someembodiments, chamber enclosure 5020 ideally rotates only once whencollecting samples during each usage of ingestible device 5010 so thataccess port 5024 aligns with each of chamber openings 5022 serially andonly once. That is, during collection of samples, access port 2224 doesnot bypass any chamber opening 5022 and also does not return to aprevious chamber opening 5022 during its rotation.

In some embodiments, chamber enclosure 5020 can rotate in abidirectional motion before completing one revolution and/or performmultiple revolutions during one usage of the ingestible device 5010 sothat at least one chamber opening 5022 is exposed multiple times. Achamber opening 5022 may need to be exposed multiple times if itscorresponding chamber stores solids or semi-solid reagents, sensors orcleaning agents for cleaning the GI tract.

As illustrated in FIG. 33A, shown therein generally is ingestible device5010 in an open position 5010 a in which access port 5024 on chamberenclosure 5020 is aligned with a chamber opening 5022. In thisconfiguration, ingestible device 5010 may collect substances throughchamber opening 5022. In other words, the contents of the GI tract maybe forced into exposed chamber 5018 through muscular contractions (e.g.,peristalsis).

Thereafter, chamber enclosure 5020 may rotate to seal chamber opening5022. FIG. 33B shows ingestible device 5010 with a partiallyopen/partially closed position 5010 b in which access port 5024 has beenrotated such that chamber enclosure 5020 partially seals chamber opening5022.

FIG. 33C shows ingestible device 5010 in a closed position 5010 c, inwhich the chamber enclosure 5020 has been rotated a distance such thataccess port 5024 completely seals chamber opening 5022. If chamberenclosure 5020 has not rotated one revolution, chamber enclosure 5020may continue to rotate in the same direction in order to align accessport 5024 with another chamber opening 5022 depending if ingestibledevice 5010 has been configured to perform another operation (i.e.sampling or distribution).

In another example embodiment, chamber enclosure 5020 may be stationaryand storage sub-unit 5016 may instead rotate to align its one or morechamber openings 5022 with access port 5024. Rotating storage sub-unit5016 instead of chamber enclosure 5020 may provide greater control overthe rotation motion and a more constant motion since storage sub-unit5016 would not be subjected to a varying viscosity arising from thecontents in the GI tract. This arrangement, however, may limit a volumeof at least one of chambers 5018.

In some embodiments, chamber enclosure 5020 or storage sub-unit 5016 mayrotate in a predetermined sequence of bidirectional rotational motions.As described above, when storage sub-unit 5016 is configured to rotateinstead of chamber enclosure 5020, the volume of at least one ofchambers 5018 can be limited. In order to avoid having to limit thevolume of the chambers 5018, non-recess areas that may be used toseparate different chambers 5018 in storage sub-unit 5016 may beminimized in volume or removed. Ingestible device 5010 can rotate in afirst direction for aligning access port 5024 with one of the twoadjacent chambers. Ingestible device 5010 can be configured to rotate ina second direction that is opposite to the first direction in order toavoid cross contamination between samples collected into or substancesreleased from those two adjacent chambers.

Ingestible device 5010 may be used for collecting usable samples fromthe contents of the GI tract (e.g., 100 μL sized samples) andmaintaining each sample in isolation from one another until the samplesare extracted.

In some embodiments, ingestible device 5010 may also be configured toconduct in-vivo measurements. Ingestible device 5010 is introduced intothe body with some of chambers 5018 being empty and some of chambers5018 carrying at least one reagents. At a predefined location in thebody, ingestible device 5010 is configured to collect a sample from theGI tract and to store the sample into a chamber carrying at least onereagent. After collection, in-vivo analysis may be conducted based onhow the collected sample interacts with the reagent inside chamber 5018.For example, ingestible device 5010 may use a biochemistry assay, suchas an enzyme-linked immunosorbent assay (ELISA), for performing in-situexperiments on collected samples. Alternatively, peripherals can beincluded into chambers 5018 for changing the dynamics of several in-vivoanalysis and measurements. The peripherals may include a light source, areceiver, a transducer, a heater, and the like. In general, the in-vivoexperiments vary according to the type of information that is beingsought.

FIG. 34 illustrates an exploded view of the components of ingestibledevice 5010 in one example embodiment. First portion 5012 of ingestibledevice 5010 includes an end closure 5030, and electronic componentsembedded on a main printed circuit board (PCB) 5032 including acommunication subsystem having communication peripherals 5034 and atransceiver 5036, a main microcontroller (i.e. processor) 5038, a powersupply 5040 and other peripheral components described in further detailbelow. Second portion 5014 of ingestible device 5010 generally includesa motor 5042, storage sub-unit 5016, a secondary PCB 5044, an encodingmagnet arrangement 5046 m and the chamber enclosure 5020. Generally, byplacing main PCB 5032 and secondary PCB 5044 in distinct regions insideingestible device 5010, they may be prevented from experiencing the sameelectrical or physical hazards. Motor 42 is inserted into a motorcompartment 5054 that is located in the center of storage sub-unit 5016.PCB 5044 is annular and includes one or more peripheral electroniccomponents (e.g., a capacitor 5062 and a resistor 4060, which can beused as a pull-up resistor), and a sensor 5064. 5039 is a magneticswitch. 5042 s is a shaft. 5056 are access holes.

End enclosure 5030 provides a hollow space defined by an inner wall 5048that is cylindrical with a domed end portion. End enclosure 5030 alsoincludes engagement members 5050 for aligning and releasably engagingwith storage sub-unit 5016 to releasably lock end enclosure 5030 inplace during operation. In particular, engagement members 5050releasably engage complementary structures 5052 in storage sub-unit5016. When end enclosure 5030 locks with storage sub-unit 5016, endenclosure 5030 overlaps with a rear of storage sub-unit 5016 and createsa seal. In some embodiments, the overlap between end enclosure 5030 andstorage sub-unit 5016 may span a width of 3 millimeters.

Some or all of the sponges of the above-described sampling systems maycontain one or more preservatives (see discussion above). Typically, theassay sponge and/or the volume sponge 1230 and/or the transfer spongecontain one or more preservatives. Typically, the preservative(s) areselected based on the analyte of interest, e.g., an analyte (such as anucleic acid or protein biomarker) for a GI disorder.

Examples of such GI orders include inflammatory bowel disease, Crohn'sdisease (e.g., active Crohn's disease, refractory Crohn's disease, orfistulizing Crohn's disease), ulcerative colitis, indeterminate colitis,infectious colitis, microscopic colitis, drug or chemical-inducedcolitis, diverticulitis, ischemic colitis, pseudomembranous colitis,hemorrhagic colitis, hemolytic-uremic syndrome colitis, collagenouscolitis, colitis associated with disorders of innate immunity as inleukocyte adhesion deficiency-1, gastritis, peptic ulcers, stressulcers, bleeding ulcers, gastric hyperacidity, dyspepsia, gastroparesis,Zollinger-Ellison syndrome, gastroesophageal reflux disease, short-bowel(anastomosis) syndrome, mucositis (e.g., oral mucositis,gastrointestinal mucositis, nasal mucositis and proctitis), necrotizingenterocolitis, esophagitis, a hypersecretory state associated withsystemic mastocytosis, basophilic leukemia, hyperhistaminemia, Celiacdisease (e.g., nontropical Sprue), enteropathy associated withseronegative arthropathies, chronic granulomatous disease, foodallergies, enterocolitis (e.g., Helicobacter pylori-infected chronicactive gastritis), other forms of gastrointestinal inflammation causedby an infectious agent, irritable colon syndrome, small intestinalbacterial overgrowth (SIBO) and pouchitis. “Inflammatory Bowel Disease”or “IBD” is a chronic inflammatory autoimmune condition of thegastrointestinal (GI) tract. Although the cause of IBD remains unknown,several factors such as genetic, infectious and immunologicsusceptibility have been implicated. IBD is much more common inCaucasians, especially those of Jewish descent. A chronic inflammatoryautoimmune condition of the gastrointestinal (GI) tract presentsclinically as either ulcerative colitis (UC) or Crohn's disease (CD).Both IBD conditions are associated with an increased risk for malignancyof the GI tract. “Crohn's disease” (“CD”) is a chronic transmuralinflammatory disease with the potential to affect any part of the entireGI tract, and UC is a mucosal inflammation of the colon. Both conditionsare characterized clinically by frequent bowel motions, malnutrition,and dehydration, with disruption in the activities of daily living. CDis frequently complicated by the development of malabsorption,strictures, and fistulae and may involve repeated surgery. UC, lessfrequently, may be complicated by severe bloody diarrhea and toxicmegacolon, also involving surgery. The most prominent feature Crohn'sdisease is the granular, reddish-purple edematous thickening of thebowel wall. With the development of inflammation, these granulomas oftenlose their circumscribed borders and integrate with the surroundingtissue. Diarrhea and obstruction of the bowel are the predominantclinical features. As with ulcerative colitis, the course of Crohn'sdisease may be continuous or relapsing, mild or severe, but unlikeulcerative colitis, Crohn's disease is not curable by resection of theinvolved segment of bowel. Most patients with Crohn's disease involvesurgery at some point, but subsequent relapse is common and continuousmedical treatment is usual. Crohn's disease may involve any part of thealimentary tract from the mouth to the anus, although typically itappears in the ileocolic, small-intestinal or colonic-anorectal regions.Histopathologically, the disease manifests by discontinuousgranulomatomas, crypt abscesses, fissures and aphthous ulcers. Theinflammatory infiltrate is mixed, consisting of lymphocytes (both T andB cells), plasma cells, macrophages, and neutrophils. There is adisproportionate increase in IgM- and IgG-secreting plasma cells,macrophages and neutrophils. “Ulcerative colitis (UC)” afflicts thelarge intestine. The course of the disease may be continuous orrelapsing, mild or severe. The earliest lesion is an inflammatoryinfiltration with abscess formation at the base of the crypts ofLieberkuhn. Coalescence of these distended and ruptured crypts tends toseparate the overlying mucosa from its blood supply, leading toulceration. Symptoms of the disease include cramping, lower abdominalpain, rectal bleeding, and frequent, loose discharges consisting mainlyof blood, pus and mucus with scanty fecal particles. A total colectomymay be involved for acute, severe or chronic, unremitting ulcerativecolitis. A “symptom” of a disease or disorder (e.g., inflammatory boweldisease, e.g., ulcerative colitis or Crohn's disease) is any morbidphenomenon or departure from the normal in structure, function, orsensation, experienced by a subject and indicative of disease.

Biomarkers

A biomarker as described herein can be a nucleic acid, such as DNA orRNA, a protein, a small molecule, or a bacterium. Biomarkers asdescribed herein are present in the GI tract, and can be used to detecta disease in the GI tract of a subject, e.g., an inflammatory disease.Biomarkers can also be used to monitor the progress or remission of adisease of the GI tract, e.g., an inflammatory disease. The biomarkerscan be collected from any portion of the GI tract, e.g., the biomarkerscan be collected form the distal small bowel to the proximal large bowelof a subject. In some embodiments, the biomarkers are produced by thecells of the subject in the GI tract. In some embodiments, thebiomarkers are produced by a microorganism, e.g., a bacterium, in the GItract. In exemplary embodiments, the biomarkers described herein arepresent or produced in the intestinal mucosa, thereby indicatingintestinal inflammation.

In some embodiments, the biomarker is a small molecule. A small moleculeis an organic compound of low molecule weight (≦100 daltons,approximately). The conditions within an ingestible device, as disclosedherein, can be formulated to be improve the capacity of the device toretain small molecules. In some embodiments, one or more small moleculesenter a device as disclosed herein in the GI tract, and are contactedwith one or more preservatives, e.g., a mixture of preservatives thatstabilize small molecules and inhibit their degradation. In someembodiments, the small molecule biomarker is a small molecule drug,e.g., a small molecule drug used to treat an inflammatory disease. Insome embodiments, the small molecule biomarker is cyclosporine.

In some embodiments, the biomarker is a nucleic acid. In someembodiments, one or more nucleic acid molecules enter a device asdisclosed herein in the GI tract, and are contacted with one or morenucleic acid preservatives, e.g., a mixture of preservatives thatstabilize nucleic acids and inhibit their degradation. In someembodiments, the nucleic acid is a DNA molecule. In some embodiments,the nucleic acid is an RNA molecule. There are many types of nucleicacid molecules known in the art that can be a biomarker, as describedherein. In some embodiments, the nucleic acid biomarker can be a mRNA, asingle-stranded RNA, a double-stranded RNA, an antisense RNA, a siRNA, amiRNA, a piRNA, a lincRNA, a tRNA, a ribozyme, a ribosomal RNA, or asnoRNA.

In some embodiments, the nucleic acid biomarker is a nucleic acid drug,e.g., a nucleic acid drug used to treat an inflammatory disease. In someembodiments, the nucleic acid biomarker is mongersen. Mongersen is aSMAD7 antisense oligonucleotide that can be used to treat inflammatorydiseases of the GI tract, e.g., Crohn's disease and IBD.

Nucleic acid preservatives can be used to prevent or reduce the rate ofnucleic acid degradation or denaturation, and/or increase the stabilityof nucleic acids, e.g., to maintain nucleic acid structure. In someembodiments, the nucleic acid preservative is nuclease inhibitor(deoxyribonuclease inhibitor). In some embodiments, the nucleic acidpreservative is a ribonuclease inhibitor. Nuclease inhibitors andribonuclease inhibitors are known in the art, and have been describedin, e.g., U.S. Pat. No. 6,224,379, herein incorporated by reference inits entirety. In some embodiments, the nucleic acid preservative mixturecan include EDTA, sodium citrate, an ammonium sulphate. In someembodiments, the RNA preservative mixture includes 2 mL of 0.5M EDTA,1.25 ml of 1 M sodium citrate, 35 g of ammonium sulphate, and 46.8 mL ofdH20. In some embodiments, the RNA preservative is an RNAlater™stabilization solution (ThermoFisher Scientific), as described in U.S.Pat. No. 7,056,673, which is herein incorporated by reference in itsentirety. In some embodiments, an RNA preservative can include one ormore of triphenylmethane dyes (such as methyl green, crystal violet,pararosaniline, or tris-(4-aminophenyl)methane), cresyl violet,polyamines, and cobalt ions. In some embodiments, an RNA preservativecan include one or more of spermine, spermidine,1,10-diamino-4,7-diazadecane, 1,11-diamino-4,8-diazaundecane,1,13-diamino-4,10-diazatridecane, 1,14-diamino-4,11-diazatetradecane,1,15-diamino-4,12-diazapentadecane, 1,16-diamino-4,13-diazahexadecane,1,17-diamino-4,14-diazaheptadecane, 1,18-diamino-4,15-diazanonadecane,1,19-diamino-4,16-diazaeicosane, and 1,20-diamino-4,17-diazaheneicosane.

In some embodiments, the biomarker is a protein. In some embodiments,one or more proteins enter a device as disclosed herein in the GI tract,and are contacted with one or more preservatives, e.g., a mixture ofpreservatives that stabilize proteins and inhibit their degradation.

In some embodiments, the protein biomarker is a cytokine. The term“cytokines” refers to a broad group of secreted small proteins,typically less than 30 kD, that are involved in cell signaling in avariety of cellular and tissue contexts. Many cytokines modulate theimmune system and are involved in inflammation and autoimmune disease.Cytokines can have pro-inflammatory or anti-inflammatory functions.Cytokines are produced by many different cell types, including immunecells, such as, e.g., macrophages, lymphocytes (e.g., B lymphocytes, Tlymphocytes), monocytes, mast cells, endothelial cells, fibroblasts, Thelper cells, and stromal cells. Exemplary types of cytokines include,without limitation, chemokines, interferons, interleukins, lymphokines,monokines, and tumor necrosis factors.

In some embodiments, the protein biomarker is a chemokine. Chemokinesare chemotactic cytokines that can function to regulate the immunesystem by inducing chemotaxis or chemokinesis in immune cells duringinflammation, e.g., leukocytes, monocytes, macrophages, T lymphocytes,mast cells, eosinophils, and neutrophils. For example, chemokines canactivate and mobilize white blood cells in acute and chronicinflammation. Chemokines can be divided into four classes: alpha (CXC),beta (CC), gamma (C), and delta (CX3C). Chemokines can include, but arenot limited to, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8,CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17,CCL1, CCL2, CCL3, CCL3L1, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9/CCL10,CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20,CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, XCL1, XCL2,XCL2, and CX3CL1.

Chemokine receptors include CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CCR1,CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, CX3CR1,and DARC. In some embodiments, the protein biomarker is a chemokine ofthe alpha, beta, gamma or delta family. In some embodiments, the proteinbiomarker is CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8,CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17,CCL1, CCL2, CCL3, CCL3L1, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9/CCL10,CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20,CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, XCL1, XCL2,XCL2, or CX3CL1. In some embodiments, the protein biomarker is areceptor that interacts with a chemokine, i.e., a chemokine receptor.

In some embodiments, the protein biomarker is an interferon (IFN).

Interferons are proteins produced by a variety of cells, e.g., such as Tcells and fibroblasts, to regulate the immune system in response toinfection or cancerous cells. IFNs are divided into three classes: typeI, type II, and type III IFNs. Interferons can also be classified asalpha, beta, gamma, tau, or omega interferons. Interferons can include,but are not limited to, IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8,IFNA10, IFN13, IFNA14, IFNA16, IFNA17, IFNA21, IFNG, IFNB1, IFNW, IFNE1,and IFNK. In some embodiments, the protein biomarker is a type I, typeII, or type III interferon. In some embodiments, the protein biomarkeris a, alpha, beta, gamma, tau, or omega interferon. In some embodiments,the protein biomarker is IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7,IFNA8, IFNA10, IFN13, IFNA14, IFNA16, IFNA17, IFNA21, IFNG, IFNB1, IFNW,IFNE1, or IFNK. In some embodiments, the protein biomarker can beinterferon-γ.

In some embodiments, the protein biomarker is an interleukin.Interleukins modulate the immune system, and are involved in a widevariety of biological process, such as promoting the development anddifferentiation of T and B lymphocytes and hematopoietic cells.Interleukins are produced by many different cell types, including immunecells, such as, for example, leukocytes, T lymphocytes, e.g., CD4 Tlymphocytes, monocytes, macrophages, and endothelial cells. Interleukinscan be divided into families, including the interleukin 1, interleukin2, interleukin 3, interleukin 4, interleukin 5, interleukin 6,interleukin 7 and 9, interleukin 8, interleukin 10, interleukin 11,interleukin 12, interleukin 13, interleukin 15, and interleukin 17families. In some embodiments, the protein biomarker is a protein ofinterleukin family 1, 2, 3, 4, 5, 6, 7 and 9, 8, 10, 11, 12, 13, 15, or17. Interleukins can include, but are not limited to, IL-1, IL-2, IL-3,IL-4, IL-5, IL-6, IL-7, IL-8 (CLCL8), IL-9, IL-10, IL-11, IL-12, IL-13,IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23,IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33,IL-34, IL-35, IL-36 and IL-37. In some embodiments, the proteinbiomarker is a protein of interleukin family 1, 2, 3, 4, 5, 6, 7 and 9,8, 10, 11, 12, 13, 15, or 17. In some embodiments, the protein biomarkeris IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8 (CLCL8), IL-9, IL-10,IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20,IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30,IL-31, IL-32, IL-33, IL-34, IL-35, IL-36 or IL-37.

In some embodiments, the protein biomarker is a lymphokine. Lymphokinesare produced and secreted by lymphocytes, such as T cells, whenlymphocytes contact antigens. Lymphokines regulate the immune response,and are involved in, e.g., attracting and activating immune cells, e.g.,macrophages, lymphocyte transformation, and cell-mediated immunity.Lymphokines include, but are not limited to IL-2, IL-3, IL-4, IL-5,IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), andinterferon-γ. In some embodiments, the protein biomarker is GM-CSF.

In some embodiments, the protein biomarker is a tumor necrosis factor.The tumor necrosis factor (TNF) superfamily of cytokines are involved ina large variety of biological processes, including immune regulation andapoptosis. Tumor necrosis factors include, but are not limited to, TNFalpha (TNF, TNF α), lymphotoxin-alpha (LTA, LT-alpha, TNF-β),lymphotoxin-beta, (LTB, LT-beta, TNFC), CD40 ligand (CD40L, gp39,TNFSF7), CD70 (CD27L, TNFSF7), TNFSF4 (OX40L), TNFSF8 (CD30L), Fasligand (FASL, FASLG), extodysplasin A (EDA), TNFSF9 (4-1BBL),TNF-related apoptosis inducing ligand (TNFSF10, TRAIL), TNFSF11,TNFSF12, TNFSF13, TNFSF13B, TNFSF14, TNFSF15, and TNFSF18. In someembodiments, the protein biomarker is TNF alpha (TNF, TNF α),lymphotoxin-alpha (LTA, LT-alpha, TNF-β), lymphotoxin-beta, (LTB,LT-beta, TNFC), CD40 ligand (CD40L, gp39, TNFSF7), CD70 (CD27L, TNFSF7),TNFSF4 (OX40L), TNFSF8 (CD30L), Fas ligand (FASL, FASLG), extodysplasinA (EDA), TNFSF9 (4-1BBL), TNF-related apoptosis inducing ligand(TNFSF10, TRAIL), TNFSF11, TNFSF12, TNFSF13, TNFSF13B, TNFSF14, TNFSF15,or TNFSF18.

In some embodiments, the protein biomarker is an integrin or a ligand toan integrin. In some embodiments, the protein biomarker is α4β7integrin. In some embodiments, the protein biomarker is mucosaladdressin cell adhesion molecule-1 (MAdCAM-1). The β1 integrin competeswith β7 integrin for binding to α4 integrin on T cells. Expression ofα4β7 integrin on T cells promotes the preferential trafficking of Tcells to sites in the intestines, such as Peyer's patches. α4β7 integrinbinds to the mucosal vascular addressin MAdCAM-1, which helps to directleukocytes, such as T cells, into mucosa of the GI tract. MadCAM isexpressed specifically on the venules of the mesenteric lymph node inthe intestinal walls, and in Peyer's patches (PP). MadCAM s unregulatedon the intestinal venules during inflammation.

In some embodiments, the protein biomarker is a monokine. Monokines areproduced by immune cells, such as macrophages and monocytes, and helpmediate the immune system, e.g., by attracting neutrophils viachemotaxis.

In some embodiments, the protein biomarker is an immunoglobulin. In someembodiments, the protein biomarker is an autoantibody associated with anautoimmune or inflammatory disease, e.g., an autoantibody associatedwith celiac disease, such as, but not limited to, tissuetransglutaminase, gliadin, and endomysial antibodies. In someembodiments, the protein biomarker is anti-tissue transglutaminaseantibody (tTG). Tissue transglutaminase is an enzyme that is abundant inthe endothelial cells of the small intestines. The abnormal activationor dysregulation of tissue transglutaminase is associated with diseasessuch as celiac disease and inflammatory disorders. Anti-tissuetransglutaminase antibodies are present in subjects who are allergic todietary gluten. In some embodiments, the protein biomarker isanti-gliadin antibody (GP). Gliadin is a prolamin that is found inwheat, and is a component of gluten that can be antigenic to subjectswith a celiac disease or a gluten allergy. Gliadins can be classified asα gliadin, β gliadin, or γ gliadin. IgA, IgG, or IgE autoantibodies canbe produced that bind to each type of gliadin in subjects with celiacdisease or with a gluten sensitivity. In some embodiments, the proteinbiomarker is anto-endomysial antibody (EMA). The presence of EMA IgAantibodies correlates with gluten-sensitivity, celiac disease, anddermatitis herepeiformis.

In some embodiments, the protein biomarker is a protein or peptide drug,e.g., a protein or peptide drug used to treat an inflammatory disease.In some embodiments, the protein biomarker is a therapeutic antibody orother protein that binds to a protein involved in inflammatory disease,e.g., a therapeutic antibody that targets a cytokine. In someembodiments, the protein biomarker is a therapeutic antibody or otherprotein that targets and binds to tumor necrosis factor alpha (TNFα). Insome embodiments, the protein biomarker is infliximab, adalimumab,certolizuman pegol, golimumab, or entanercept, or an antigen-bindingportion thereof. In some embodiments, the protein biomarker is anantibody or other protein that binds to a therapeutic antibody orantigen-binding portion thereof, e.g., an antibody that binds to atherapeutic antibody or antigen-binding portion thereof that is used totreat inflammatory or autoimmune diseases of the GI tract. In someembodiments, the protein biomarker is an antibody or other protein thatbinds to a therapeutic antibody or antigen-binding portion thereof thattargets and binds to TNFα, e.g., the protein biomarker is an antibodythat binds to infliximab, adalimumab, certolizuman pegol, golimumab, orentanercept, or an antigen-binding portion thereof.

In some embodiments, the protein biomarker is secretory IgA. SecretoryIgA is the predominant immunoglobulin isotype present in mucosalsecretions, and is important for maintaining the immune barrier in thegastrointestinal tract. Secretory IgA helps to control the intestinalmilieu in response to bacteria, parasites, and viruses. Elevated levelsof fecal secretory IgA are associated with an upregulated immuneresponse in the GI tract, and can therefore indicate inflammation.

In some embodiments, the protein biomarker is a protein that is not animmunoglobulin (e.g., not an antibody or autoantibody).

In some embodiments, the protein biomarker is a fecal biomarker. Fecalbiomarkers are known in the art, see, e.g., Lehmann et al., Ther. Adv.Gastroenterol., 8(1):23-26, 2015, herein incorporated by reference inits entirety. In some embodiments, the protein biomarker is produced andsecreted by neutrophils in the intestinal mucosa in response togastrointestinal inflammation, e.g., inflammation caused by IBD.

In some embodiments, the protein biomarker is calprotectin. Inflammationcaused by IBD results in an influx of neutrophils to the intestinalmucosa of the gastrointestinal tract. Calprotectin is a 24 kDa dimer ofcalcium binding proteins S100A8 and S100A9. Calprotectin is apro-inflammatory protein, and the concentration of calprotectin isproportional to the intensity of neutrophils in the gut mucosa. Elevatedlevels of faecal calprotectin indicates the migration of neutrophils tothe intestinal mucosa, and can serve as a marker for intestinalinflammation caused by, for example, IBD, Crohn's Disease, or ulcerativecolitis (see Lehmann et al., Ther. Adv. Gastroenterol., 8(1):23-26,2015). Elevated levels of calprotectin can also be used to differentiatebetween IBD and IBS.

In some embodiments, the protein biomarker is S100A12. S100A12 is aspecific neutrophilic protein that is upregulated during active IBD.Release of S100A12 from intestinal mucosa correlates with inflammation,and fecal levels of S100A12 can be used to diagnose IBD.

In some embodiments, the protein biomarker is lactoferrin. Lactoferrinis an iron-binding protein expressed by activated neutrophils, andmucosal epithelial cells. Elevated levels of faecal lactoferrin isindicative of inflammation in the gastrointestinal system caused by,e.g., chronic IBD, ulcerative colitis, and Crohn's disease (see Kane etal., Am J Gastroenterol. 98(6):1309-14, 2003; Lehmann et al., Ther. Adv.Gastroenterol., 8(1):23-26, 2015).

In some embodiments, the protein biomarker is M2-pyruvate kinase (M2PK).M2PK is a multifunctional protein that is present in undifferentiatedand proliferating tissues. Fecal M2PK levels are increased in activeIBD, and M2PK has been shown to be capable of differentiating betweenIBD and IBS.

In some embodiments, the protein biomarker is neopterin. Neopterin is anintermediate metabolite of biopterin that is released from macrophages.Levels of neopterin are higher in active IBD than in inactive disease,and neopterin concentration levels correlate with mucosal lesionseverity.

In some embodiments, the protein biomarker is a metalloproteinase (MMP).Metalloproteinases belong to a family of zinc-dependent endopeptidases.MMPs such as MMP-9 are secreted by activated neutrophils in IBD, and inulcerative colitis biopsies, MMP-1, MMP-2, MMP-3, and MMP-9concentrations are elevated.

In some embodiments, the protein biomarker is a myeloperoxidase (MPO).Myeloperoxidases are lysosomal proteins that are released by activatedneutrophils during inflammation.

In some embodiments, the protein biomarker is a polymorphonuclearelastase (PMN elasetase). PMN elastase is released by activatedneutrophils, and subjects with active IBD have higher concentrations offecal PMN elastase than subjects with IBS or inactive IBD.

In some embodiments, the protein biomarker is alpha 1 antitrypsin (A1A).A1A is a linear glycoprotein predominantly synthesized in the liver, butis also made by intestinal macrophages, monocytes and epithelial cells.A1A is resistant to degradation in the gut, and is a marker forintestinal protein loss and permeability. A1A concentration levels havebeen shown to be useful for evaluating and monitoring chronicinflammatory intestinal diseases.

In some embodiments, the protein biomarker is eosinophilic protein X(EPX). EPX is released from activated eosinophils, including in thegastrointestinal tract.

Protein preservatives can be used to prevent or reduce the rate ofprotein degradation or denaturation, and/or increase the stability ofproteins, e.g., to maintain protein structure. Preservatives caninclude, by way of example, protease inhibitors, surfactants (e.g.,nonionic surfactants), emulsifiers, acids, parabens, esters and proteinstabilizers.

In some embodiments, the preservative can prevent or reduce thedigestion or degradation of proteins by one or more proteases. In someembodiments, the preservative can be a protease inhibitor. In someembodiments, the protease inhibitor is a serine protease inhibitor, ametalloprotease inhibitor, an aminopeptidase inhibitor, a cysteinepeptidase inhibitor, or an aspartyl protease inhibitor. In someembodiments, the protease inhibitor can prevent or reduce digestion byproteases such as, but not limited to, trypsin, chymotrypsin, plasminkallikrein, thrombin, papain, cathepsin B, cathepsin L, calpain andstaphopain, endoproteinase Lys-C, Kallikrein, and thrombin. In someembodiments, the protease inhibitor can be4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF, CAS30827-99-7), aprotinin (CAS 9087-70-1), bestatin (CAS 58970-76-6), E-64(CAS 66701-25-5), leupeptin (CAS 103476-89-7), pepstatin A (CAS26305-03-3), or N-p-Tosyl-L-phenylalanine chloromethyl ketone (TPCK). Insome embodiments, the protein biomarker preservative includes4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF, CAS30827-99-7), aprotinin (CAS 9087-70-1), bestatin (CAS 58970-76-6), E-64(CAS 66701-25-5), leupeptin (CAS 103476-89-7), pepstatin A (CAS26305-03-3), DMSA, and bovine serum albumin, and, optionally,N-p-Tosyl-L-phenylalanine chloromethyl ketone (TPCK).

In some embodiments, the preservative can be a protein stabilizer suchas, for example, Trehalose or Dextran.

A preservative as disclosed herein can be an acid. In some embodiments,the preservative can be an acid with a pKa between 3 and 7. In someembodiments, the preservative can be citric acid, or sorbic acid.

In some embodiments, the preservative can be a surfactant such as apolysorbate. Exemplary polysorbates include, for example, polysorbate 20(polyoxyethylene (20) sorbitan monolaurate), polysorbate 40(polyoxyethylene (20) sorbitan monopalmitate), polysorbate 60(polyoxyethylene (20) sorbitan monostearate), polysorbate 80(polyoxyethylene (20) sorbitan monooleate), sorbitan monolaurate,sorbitan monopalmitate, sorbitan monostearate, sorbitan tristearate, andsorbitan monooleate.

In some embodiments, the preservative is a paraben, parahydroxybenzoate,or ester of parahydroxybenzoic acid (4-hydroxybenzoic acid). In someembodiments, the preservative can be propyl paraben.

In some embodiments, the preservative can include dimethyl sulfoxide(DMSA). In some embodiments, the preservative can include bovine serumalbumin.

The preservative can be a mixture of two or more of a proteaseinhibitor, a surfactant, an emulsifier, an acid, a paraben, and anester. For example, a preservative as described herein can include amixture of two or more protease inhibitors. In some embodiments, apreservative as described herein can include a mixture of one or moreprotease inhibitors, and one or more acids. In some embodiments, apreservative as described herein can include a mixture of one or moreprotease inhibitors, one or more acids, and an ester, e.g., a paraben.In some embodiments, a preservative as described herein can include amixture of one or more protease inhibitors, one or more acids, one ormore esters, and one or more surfactants. In some embodiments, thepreservative can include the HALT™ protease inhibitor cocktail (ThermoFisher). In some embodiments, the preservative can include the HALT™protease inhibitor cocktail (Thermo Fisher) and TPCK. In someembodiments, the preservative can be bactericidal to preserve a proteinbiomarker. In some embodiments, the preservative mixture that isbactericidal includes citric acid (CAS 77-92-9), sorbic acid (CAS110-44-1), propylparaben (CAS 94-13-3), tween 80 (CAS 9005-65-6),ethanol, bovine serum albumin, and TPCK (CAS 402-71-1).

In some embodiments, a preservative mixture containing one or moreprotease inhibitors can be contacted with a protein in thegastrointestinal tract to stabilize the protein. In some embodiments,the protein is an immunoglobulin. In some embodiments, the protein is anIgA or IgM. In some embodiments, the protein is a secretory IgA. In anexemplary embodiment, a preservative mixture containing AEBSF,aprotinin, bestatin, E-64, leupeptin and pepstatin A protease inhibitors(HALT™, Thermo Fisher), and N-p-Tosyl-L-phenylalanine chloromethylketone (TPCK, Sigma Aldrich) can be used to stabilize one or moreimmunoglobulin proteins in the gastrointestinal tract, e.g., secretoryIgA.

In some embodiments, a preservative mixture containing one or moreprotease inhibitors, acids, parabens, and surfactants can be contactedwith a protein in the gastrointestinal tract to stabilize the protein.In some embodiments, the protein is not an immunoglobulin. In anexemplary embodiment, a preservative mixture containing AEBSF,aprotinin, bestatin, E-64, leupeptin and pepstatin A protease inhibitors(HALT™, Thermo Fisher), N-p-Tosyl-L-phenylalanine chloromethyl ketone(TPCK, Sigma Aldrich), citric acid, sorbic acid, propyl paraben,polysorbate 80 (Tween 80), BSA can be used to stabilize one or morenon-immunoglobulin proteins in the gastrointestinal tract, e.g., acytokine, calprotectin, S100A12, lactoferrin, M2-pyruvate kinase,neopterin, a metalloproteinase, a myeloperoxidase, polymorphonuclearelastase, and/or alpha 1 antitrypsin eosinophilic protein X.

In some embodiments, one or more internal controls are included in aningestible device, as described herein, that is used to collect one ormore biomarker analytes. The internal control can be used to monitor thestability and degradation of small molecules, nucleic acids, and/orproteins in the device over time. In some embodiments, the internalcontrol can be a small molecule, a nucleic acid, and/or a protein. Insome embodiments, the small molecule internal control can be 2,4dinitrophenol (2,4, DNP), femocene, and/or a deuterium-labeledcholesterol. In some embodiments, the nucleic acid internal control canbe a DNA internal control. In some embodiments, the nucleic acidinternal control can be a RNA internal control. In some embodiments, theRNA internal control can be a G+C-rich (60%) RNA molecule with extensivesecondary structure, based on a modified delta virus genome, asdescribed in Dingle et al., J. Clin. Microbiol. 42(3):1003-1011, 2004,herein incorporated by reference in its entirety. In some embodiments,the protein internal control can be human serum albumin (HAS),fluorescein isothiocyanate, and/or biotin.

Microbial Preservatives

The devices and methods disclosed herein can also be used to collect asample of microbial cells, e.g., bacterial cells, in thegastrointestinal (GI) tract of a subject, and the sampled cells can beanalyzed to identify and quantify the cells. In some embodiments, theingestible devices and methods disclosed herein use one or morepreservatives that stabilize bacterial cells collected in the device, sothat once the device exits the subject's body, an accurate assessmentcan be made as to the identity and number of bacteria present at thelocation of the GI tract where the bacteria were collected. Theingestible devices and methods disclosed herein can providerepresentative microbiome data at the site of bacterial cell collectionin the GI tract.

Any microbe present in the gastrointestinal tract can enter aningestible device as described herein. The microbe can be a bacterium, afungus, or a protest. In exemplary embodiments, at least one bacteriumenters an ingestible device as described herein. In some embodiments,the microbe is part of the normal microflora of the gastrointestinaltract. In some embodiments, analysis of the microbes that have collectedin the ingestible device can provide information about the microflora ofthe gastrointestinal tract that can be predictive about the health ofthe gastrointestinal tract, and/or diagnose or predict a disorder of thegastrointestinal tract. For example, information about the abundance ofcertain types of bacteria relative to the bacteria present in a controlsample, e.g., a sample from a subject with a healthy gastrointestinaltract, can be used to diagnose or predict a disorder, such as aninflammatory disorder. In some embodiments, a taxonomic shift inbacteria, or change in the abundance of certain types of bacteria,relative to the microbiome of a healthy subject can be used to predict adisease or disorder of the gastrointestinal tract, e.g., Crohn'sdisease, inflammatory bowel disease, ulcerative colitis, irritablebowled syndrome, or small intestinal bacterial overgrowth (see, e.g.,Wright et al., Inflamm. Bowel Dis. 21(6):1219-1228, 2015; Kostic et al.,Gastroenterology 146(6):1489-1499, 2014; Sartor and Mazmanian, Am. J.Gastroenterol. Suppl. 1:15-21, 2012). In some embodiments, at least onetype of bacteria associated with an inflammatory and/or autoimmunedisease of the gastrointestinal tract can be collected in the ingestibledevice.

As used herein, the terms “stabilize” or “stabilized” means that thecells remain in the same state or in a similar state as when they werecollected in a device as described herein until the cells are lateranalyzed, such that the overall identity and number of cells has notchanged or has changed little compared to when the cells were collected.As a result, the cells collected in the device are representative of thepopulation of cells present at the site of collection, e.g., provideaccurate cell counts found at the site of collection.

The devices and methods disclosed herein overcome challenges associatedin deriving information about the microbiome based on the sampling ofbacteria in the GI tract. A significant challenge in sample analysis isthe delay that occurs between the sampling of bacteria in the GI tractusing an ingestible device, e.g., the small bowel, and the recovery ofthe device once it exits the subject's GI tract. During this period oftime, the bacterial samples are exposed to temperatures and conditionsthat facilitate the growth and multiplication of certain bacteriastrains, while eliminating of other types of bacteria. As a result, oncethe ingestible device leaves the body, certain types of bacteria in thepopulation present in the device, such as anaerobic strains, may beoverrepresented relative to other types of bacteria, and the overallnumbers of bacteria may not be representative of the population presentin the GI tract. The devices and methods disclosed herein overcome thischallenge by using preservatives to stabilize the bacteria populationcollected in the device, so that the types and numbers of bacteriapresent in the device after it exits the body are similar to the typesand numbers of bacteria that were initially collected in the device inthe GI tract.

In some embodiments, a sample of bacterial cells collected in aningestible device is contacted with a preservative that stabilizes thebacteria sample, so that the sample can provide accurate informationabout the identity and cell count of bacteria for at least 30 days afterthe sample was collected. In some embodiments, a sample of bacterialcells can be collected and stabilized an ingestible device as disclosedherein, so that the sample can provide accurate information about theidentity and cell count of bacteria for at least 1 day, 2 days, 3 days,4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28days, 29 days, or 30 days. In some embodiments, a sample of bacterialcells can be collected and stabilized in a device as disclosed herein,so that the sample can be provide accurate information about theidentity and cell count of bacteria found at the site of collectionafter the device has transited through the GI tract, and has beenrecovered and analyzed.

Bacteria can be collected by a device as described herein in anylocation within the gastrointestinal tract of a subject. In someembodiments, bacterial cells are collected in two or more locationswithin the GI tract. In some embodiments, bacterial cells are collectedin the upper gastrointestinal tract of a subject. In some embodiments,bacterial cells are collected in the large intestine. In someembodiments, the bacterial cells are collected in the small intestine.In some embodiments, bacterial cells are collected in the duodenum of asubject. In some embodiments, bacterial cells are collected in thejejunum of a subject. In some embodiments, bacterial cells are collectedin the ileum of a subject. In some embodiments, bacterial cells arecollected in the duodenum and the jejunum of a subject.

In some embodiments, a sample of bacterial cells is collected in the GItract of a subject having, or suspected of having, small intestinalbacterial overgrowth (SIBO). SIBO results from excessive bacteria in thesmall intestine. Subjects having SIBO can vary in the presentation ofdisease. Symptoms can be mild in some subjects, resulting in indigestionand bloating, to more severe, causing chronic diarrhea, weight loss, andmalabsorption. SIBO is often associated with another illness thataffects the functioning of the small intestine, including disorders thataffect the motility or movement of the small bowel, and disorders thataffect the immune system, such as, but not limited to, irritable bowelsyndrome, Crohn's disease, and achlorhydria. SIBO diagnosis involves anaccurate quantification of cells found in a sample collected from thesmall intestines. A count of more than 1×105 CFU of bacteria in a freshsample collected from the small intestines using endoscopy indicates thepresence of SIBO.

In exemplary embodiments, the preservative prevents, inhibits, orreduces the growth and/or multiplication of bacteria. In someembodiments, the preservative permanently prevents, inhibits, or reducesthe growth and/or multiplication of bacteria. In some embodiments, thepreservative is one or more of a bacteriostatic, bacteriocidal, and/orfixative compound.

Bacteriostatic preservatives arrest the growth or multiplication of thebacteria. In some embodiments, the preservative kills the bacteria,thereby preventing growth and multiplication. Bactericidal killbacteria. Bacteria enter a device as described herein in the GI tract ofa subject, and are contacted with a bacteriostatic preservative thatarrests bacterial growth and multiplication, or a bactericidalpreservative that kills the bacteria. As a result, the numbers ofbacteria in the device are representative of the bacterial microflorathat was present in the GI tract at the time the bacteria first enteredthe device.

In some embodiments, the preservative can be a bacteriostatic foodpreservative, such as, but not limited to, sorbic acid, citric acid,propyl paraben, nisin, dimethyl dicarbonate, andethylenediaminetetraacetic acid (EDTA). In some embodiments, thepreservative can be sodium azide, hydroxyurea, fusidic acid,diazolidinyl urea, imidazolidinyl urea, salicylic acid, barium andnickle chloride, metallic copper, thimerosal, 2-phenoxyethanol, orProClin™. In some embodiments, the preservative can be one or more ofsorbic acid, citric acid, propyl paraben, nisin, dimethyl dicarbonate,ethylenediaminetetraacetic acid (EDTA), sodium azide, hydroxyurea,fusidic acid, diazolidinyl urea, imidazolidinyl urea, salicylic acid,barium and nickle chloride, metallic copper, thimerosal,2-phenoxyethanol, and ProClin™.

In some embodiments, the preservative prevents or reduces nucleic aciddegradation, in addition to preventing or inhibiting the growth and/ormultiplication of bacteria. The preservation of nucleic acid integrityallows for the quantification of bacteria using PCR-based DNA or RNAanalysis methods, e.g., 16S ribosomal RNA PCR and sequencing. In someembodiments, the preservative includes EDTA.

In some embodiments, the bactericidal preservative can include one ormore of citric acid (CAS 77-92-9), sorbic acid (CAS 110-44-1),propylparaben (CAS 94-13-3), Tween 80 (CAS 9005-65-6), ethanol, bovineserum albumin, and TPCK (CAS 402-71-1). In some embodiments, thebactericidal preservative is a mixture of citric acid, sorbic acid,propyl-paraben, and Tween 80, e.g., the bactericidal preservative caninclude 2.5% (m/v) citric acid, 2.5% (m/v) sorbic acid, 2.5% (m/v)propyl-paraben), and 3.13% (m/v) Tween 80. In some embodiments, thebactericidal preservative is a mixture of sorbic acid, Tris, EDTA, Tween80, and NaCl, e.g., the bactericidal preservative can include 2.0% (m/v)sorbic acid, tris, EDTA, 1.0% (m/v) Tween 80, and 1.0% (m/v) NaCl. Insome embodiments, the bactericidal preservative is a heavy metalbactericidal mixture. In some embodiments, the bactericidal preservativeis a mixture that includes barium chloride and nickel chloride. In someembodiments, the bactericidal preservative is thimerosal, e.g., astabilizer that includes 0.1% thimerosal.

Bacterial cells collected and stabilized in an ingestible device asdisclosed herein can be analyzed using materials and methods that arewell-known in the art to identify and/or count the number of bacteria ina sample to obtain representative microbiome data at the site ofcollection. In some embodiments, 16S ribosomal RNA sequencing is used toanalyze the bacteria stabilized and collected in the apparatus ordevice. Various methods of 16S ribosomal sequencing are known in theart, and can be used to analyze bacterial samples as described herein(see, e.g., Sanschagrin and Yergeau, J. Vis. Exp. 90:51709, 2014).

In some embodiments, bacterial cells collected and stabilized in aningestible device can be counted and/or identified, and compared tobacterial cells obtained by endoscopic sampling, e.g., from smallintestine aspirates obtained by endoscopy, such as duodenal aspirates.In some embodiments, bacterial cells are collected and stabilized from asubject having SIBO or suspected of having SIBO using an ingestibledevice as described herein, and the number of bacteria in the device iscounted and compared to a control sample collected from the smallintestine of a subject through endoscopy. The control sample can be anegative control, i.e., collected from a subject who is known not tohave SIBO, or a positive control, i.e., collected from a subject knownto have SIBO. A count of more than 1×10⁵ CFU is considered SIBO whenassessed on fresh samples collected through endoscopy.

EXAMPLES Materials

Simulated Duodenal Juice (“SDJ”): SDJ was formulated by adding 2.5 mL ofa first solution (“Solution 1”) to 10 mL of a second solution (“Solution2”). Solution 1 contained nine mg Pancreatin (Sigma Aldrich Cat. No.P1750), 65 mg bovine (ox) bile (Sigma Aldrich Cat. No. B3883), and 10 mLsaline. Solution 2 contained five mg mucin (Sigma Aldrich Cat. No.M2378), and 10 mL saline. After mixing Solution 1 and Solution 2, the pHwas adjusted to 6.5 to reflect the mean pH in fasting duodenal fluidsamples. SDJ stock was aliquoted into sterile 15 mL conical tubes andfrozen at −80° C. On each day of experimentation, SDJ stock was thawedat room temperature and used within 1 hour.

HALT Protease Inhibitor Cocktail: 100×HALT™ Protease inhibitor cocktail(Thermo Fisher Cat. No. 78430).

TPCK: TPCK (Sigma Aldrich Cat. No. T4376). 30 mM stock solution in 100%ethanol.

Cidal Mix 1 (CM1) stock solution: citric acid (Sigma Aldrich Cat. No.251275), 50% stock w/v solution in water; sorbic acid (Sigma AldrichCat. No. S1626), 10% stock w/v solution in 100% ethanol; propyl paraben(Sigma Aldrich Cat. No. PHR1010), 33% stock w/v solution in 100%ethanol; and Tween® 80 (Sigma Aldrich Cat. No. P1754).

Bovine Serum Albumin (BSA): BSA (Proliant Cat. No. 7500802 Lot12G54003). 1% stock w/v in PBS or water.

Immunoglobulin Preservative Solution: 4 mL solution containing 1×HALT,20 μM TPCK in 1% BSA prepared by combining 40 μL 100×HALT, 2.66 μL 30 mMstock TPCK (Sigma Aldrich Cat. No. T4376), and 3.95 mL sterile distilledwater containing 1% BSA.

Cytokine Preservative Solution: 4 mL solution containing 1×HALT, 0.3%cidal mix, 20 μM TPCK and 1% BSA prepared by combining 24 μL citric acidstock, 120 μL sorbic acid stock, 36 μL propyl paraben, 125 μL Tween® 80,40 μL 100×HALT™, 2.66 μL 30 mM stock TPCK, and 3.65 mL sterile distilledwater containing 1% BSA.

Absorbent Material: Carwild Ivalon PVA (P4) sponge shaved to 1.3+/−0.1mm and cut to 6 mm×8.5 mm.

Protein Analytes (purified standards): Recombinant human IL6 (R&Dsystems Cat. No. 7270-IL025); IgA from human serum (Sigma Aldrich Cat.No. I1010-5MG); IgM from human colostrum (Sigma Aldrich Cat. No.18260-5MG); and FITC-HSA-Biotin (Nanocs Cat. No. HS2-BNFC).

Extraction buffers: Epitope Diagnostics Inc. (from Quantitative FecalCalprotectin ELISA kit Cat. No. KT-849); and HyCult Biotech Extractionbuffer (from Calprotectin Human ELISA kit Cat. No. HK379-02).

ELISA kits: Human IL6 Quantikine ELISA Kit (R&D systems Cat. No. D6050);Human IgA ELISA kit (Abcam Cat. No. ab196263); Human IgM ELISA kit(Abcam Cat. No. ab214568); and Internal Control ELISA Assay (Developedinternally)

Internal Control protein: FITC-HSA-Biotin (Nanocs Cat. No. HS2-BNFC).400 ug/mL working stock in filter sterilized PBS containing 0.1% BSA

Blocking reagent: SuperBlock (PBS) Blocking Buffer (Thermo Fisher CatNo. 37515)

Capture antibody: Pierce Fluorescein Isothiocyanate Antibody (ThermoFisher Cat No.: MIF2901). 10 μg/mL working stock in filter sterilizedPBS.

-   -   Assay Diluent: BSA in filter sterilized PBS at 1% w/v.    -   Detection reagent: Pierce High Sensitivity Streptavidin-HRP        (Thermo Fisher Cat No. 21130). 1:10,000 working stock in Assay        Diluent.    -   Substrate reagent: QuantaRed Enhanced Chemifluorescent HRP        Substrate (Thermo Fisher Cat No. 15159), prepared as indicated        in product literature.    -   Wash Buffer: Tween 20 at 0.01% v/v in filter sterilized PBS.    -   HABA Biotin Blocking Solution: 825 μM working stock in ethanol.

Methods

Test wells were blocked with phosphate buffered saline (PBS) containing1% BSA and plates were incubated at 4° C. for one. Wells weresubsequently washed four times with PBS containing 0.01% Tween20. Afterwashing, neat SDJ or heat-treated SDJ (both containing 1×HALT) was addedto the test wells and spiked with IgA (2500 ng/ml), IgM (1250 ng/ml), orIL6 (30 ng/ml) in 0.1% BSA. 20 μl samples were harvested immediatelyafter analyte addition. Samples were diluted 1:50 in each extractionbuffer and centrifuged at 10,000×g for 20 minutes at 4° C. For IL6detection, 100 μl supernatant was diluted 1:2 in ELISA diluent buffer.For IgA and IgM, 50 μl of extracted supernatant was diluted with 50 μlof antibody cocktail according to the IgA and IgM ELISA protocol.Experiments used neat SDJ (untreated) or heat-treated at 100° C./15 min(to eliminate heat-liable components that may hinder analyte detectionin SDJ).

Commercially available human IL6, IgA and IgM ELISA kits were used todetect these analytes according to the manufacturer's instructions.

To detect IC, an ELISA assay was developed de novo. IC was an HSAprotein conjugated to both FITC and Biotin. The rationale for using thisprotein was to capture/immobilize the IC protein via the FITC tag andthen use the Biotin tag for detection.

The following describes the preparation of an internal control standardcurve in duplicate. IC stock solution for these experiments (Nanocs CatNo. HS2-BNFC) was at 4 mg/ml. IC standard concentration range was 0 to30,000 ng/mL.

-   -   1.) Label 8 tubes: standards 1-8.    -   2.) Add 595.5 μL of 1×PBS+1% BSA into tube 1. Add 400 μL of        1×PBS+1% BSA into the remaining tubes labeled 2-8.    -   3.) Pipette 4.5 μL of 4 mg/mL stock HSA stock solution into        tube 1. This will serve as the highest concentration at 30.00        ng/mL of the standard curve. Prepare a 1:3 dilution series, as        shown in FIG. 35. Mix each tube thoroughly before the next        transfer. The 1×PBS+1% BSA solution serves as the zero standard.

The wells were coated with capture antibody as follows. The anti-FITCantibody was diluted to 10 μg/mL in PBS. 100 μL per well was plated inan ELISA plate. The plate was incubated at room temperature for 2 hourson a plate shaker set to 45 rpm or overnight at 4° C.

The wells were blocked as follows. SuperBlock blocking buffer was usedto wash and block wells. Three exchanges of 300 μL were performed,flicking the plate contents into sink and knocking the plate on a hardsurface to remove excess liquid. Incubation was not used with SuperBlockbecause blocking was immediate. SuperBlock was left in the wells untilthe samples and controls were ready for plating.

The samples and protein standards were added as follows. 100 μL ofsample (or protein standard) was pipetted into each well in duplicate.For a positive control, FITC-HSA-Biotin was diluted to 1 μg/ml into1×PBS+1% BSA. 1 ml of 0.5 μg/mL FITC-HSA-Biotin was prepared as follows.A working stock of HSA protein was prepared by diluting 1:10 of theinitial stock solution of 4 mg/mL of protein to produce 0.4 mg/ml. 2.5μL of 0.4 mg/mL HSA was added to an Eppendorf tube and the volume wasbrought up to 1 ml with 1×PBS+1% BSA. The plate was sealed and incubatedfor 2.5 hours at room temp on a plate shaker set to 45 rpm.

The plate was washed as follows. Each well was aspirated or emptied byinverting the plate and shaking the contents over a sink. The plate wasblotted against clean paper towels to remove excess liquid. The platewas washed four times by adding 300 μL of 1×PBS+0.01% Tween 20. Liquidwas completely removed at each step by blotting plate against cleanpaper towels.

Detection reagent and HABA were added as follows. Streptavidin-HRP wasdiluted in 1:10,000 in PBS+1% BSA with 20 μM HABA reagent and added 100μl per well. For a full 96-well plate, 10 ml of diluted Streptavidin-HRPwas prepared by pipetting 1 μl of Streptavidin-HRP stock solution (4.13mg/ml) and 85 μl into 10 ml of 1×PBS+1% BSA. If less detection reagentwas used, the appropriate volume was prepared for the number of samplesbeing tested. The plate was incubated for hours at room temperature on aplate shaker set to 45 rpm.

HRP substrate was added as follows. The plate was washed once with 300μl 1×PBS 0.01% Tween 20. The plate was washed three times with 300 μl1×PBS only (ensure there is no Tween 20 in the wells before addingsubstrate because it could cause a high background signal). Thesubstrate mix was made as follows. If, for example, 100 μl substrate wasadded to each well and there were about 100 wells, then this generated asolution of 10 ml. To prepare 10 ml of substrate solution, 5 ml ofEnhancer solution, 5 ml of Stable Peroxide and 100 μl of ADHP were addedto a 15 ml falcon tube. 100 μl of substrate was added and incubated for15 minutes at room temperature on a plate shaker set at 45 rpm. Areading was made on GloMax in absorbance mode at 560 nm, then onfluorescence mode Emission 580-640, Excitation Filter 520 nm. Thereaction was stopped with 10 μl stop solution and re-read, asappropriate.

The internal control ELISA reagents and equipment were as follows.

-   -   BSA (Lampire Biological Laboratories Cat No. 7500804)    -   PBS, pH 7.4 (Thermo Cat No. 10010-023)    -   Tween® 20 (Sigma Cat No. P9416)    -   Reagent grade ethanol    -   Nunc-Immuno™ MicroWell™ 96 well solid plates, MaxiSorp™ (Thermo        Fisher Cat No. 442404)    -   300 μL 8-channel pipettor    -   10 μL 8-channel pipettor    -   Single channel pipettors    -   Pipet tips    -   Reagent reservoirs    -   Light protective plate seals (TempPlate EXT Sealing Foil, USA        Scientific Cat No. 2998-7100)    -   Orbital plate shaker

The effects of varying concentrations of HABA on detection of IC proteinwere investigated using a standard concentration curve. During thedetection phase of the IC ELISA either 0, 5, 10, 20 μM HABA was mixedwith Streptavidin-HRP. As shown in FIG. 36, the IC ELISA was functionaland adding 2004 HABA improved IC signal detection.

Here, the method that was used for detecting IC after exposure to SDJ isdescribed. The impact of the presence of protein preservatives used todetect IgA, IgM and IL6 in SDJ interfered with the IC ELISA detection.The stability of IC in SDJ following incubation at 37° C. for 72 hourswas investigated.

Initially test wells were blocked with PBS containing 1% BSA to preventIC protein from binding to the plastic surface on the test plate. Plateswere incubated at 4° C. for 1 hour. Wells were subsequently washed 4×with PBS containing 0.01% Tween20. 100 μL of SDJ containing PBS only or1×HALT−/+0.15% Cidal Mix 1 in PBS containing 1% BSA was spiked with 5 μgFITC-HSA-Biotin. 20 μL samples were taken at time 0 (immediately afteranalyte addition) and again after incubation at 37° C. for 72 hours.Samples were diluted 1:50 in Epitope extraction buffer and centrifugedat 10,000×g for 20 minutes at 4° C. The supernatant was assayed for thepresence of IC by ELISA (as described above).

Results are depicted in FIG. 37. Epitope extraction buffer was effectiveat extracting IC from SDJ to enable detection of IC by ELISA. Detectionof IC by ELISA was not significantly hindered by the presence of analytepreservatives in SDJ. The IC protein was relatively stable in SDJ andcould be detected by ELISA following incubation at 37° C. for 72 hours.

Capturing and Preserving Biomarkers

A test system was established to investigate delivery of proteinpreservatives to a biorelevant surrogate matrix, which was SDJ. Atwo-step process was developed to load the absorbent material (seeabove) with preservative chemistries involved for analyte preservation.First, absorbent material was submerged in either 4 mL of ImmunoglobulinPreservative Solution, see Section 5 for the preservation of IgA and IgMproteins in SDJ, or absorbent material was submerged in 4 mL of CytokinePreservative Solution for the preservation of IL6. The absorbentmaterial was soaked in preservative solution until saturation (fiveminutes). The absorbent material was removed and dried overnight at roomtemperature in a vacuum oven. Following drying, a tripartite internalcontrol (IC) molecule including Fluorescein isothiocyanate conjugated tohuman serum albumin conjugated to Biotin (FITC-HSA-Biotin) was pipettedon top of the preservative-loaded absorbent material. Again, theabsorbent material was dried overnight at room temperature in a vacuumoven. This served as a control protein during biomarker detection. Theinternal control was used to monitor any protein degradation processthat may occur inside an ingestible as it transits the gut. In theexperiments described here, the IC was applied to each absorbentmaterial in a known amount prior to exposure to SDJ, then assayed aftervaried lengths of time thereafter. Through this method it was envisionedthat the loss of the IC could be used as a marker for general proteindegradation. The kinetics of degradation can be derived and used to backcalculate/estimate the starting concentrations of other biomarkers ofinterest.

The following experiments demonstrate effective delivery of proteinpreservatives into SDJ matrix using the absorbent material. Experimentswere set up as follows. Test wells were blocked with PBS containing 1%BSA to prevent protein analytes from binding to the plastic surface onthe test plate. Plates were incubated at 4° C. for one hour. Wells weresubsequently washed 4× with PBS containing 0.01% Tween20. The absorbentmaterial loaded with Immunoglobulin Preservative Solution was submergedin 100 μL SDJ after the SDJ was spiked with IgA (2500 ng/ml) and IgM(1250 ng/ml). The absorbent material soaked in Cytokine PreservativeSolution was submerged into SDJ either before or after spiking IL6 (1μg/mL). SDJ containing optimum preservatives (in the absence ofabsorbent material) was spiked with IgA or IgM as positive controls. 20μL samples were taken at time 0 (immediately after analyte and absorbentmaterial addition) and again after incubation at 37° C. for 18 hours.Samples were diluted 1:50 in Epitope extraction buffer and centrifugedat 10,000×g for 20 minutes at 4° C. For IL6 detection, 100 μl ofsupernatant was diluted 1:2 in ELISA diluent buffer. For IgA and IgMdetection, 50 μl of extracted sample was incubated with 50 μl of ELISAcocktail antibody according to the ELISA kit instructions.

It was determined that successful detection of IgA and IgM in SDJ wasachieved using 1% BSA and 100×HALT only, and that successful detectionof IL6 was achieved using 1% BSA, 100×HALT and 0.3% CM1.

The foregoing experiments were repeated with the 20 μL samples taken attime 0 (immediately after analyte and absorbent material addition) andagain after incubation at 37° C. for 24 hours, 48 hours and 72 hours.

FIGS. 38-43 show that: 1) the absorbent material was successfully loadedwith preservative mixes; 2) the preservative mixes were successfullydelivered to a biorelevant matrix; 3) protein biomarkers were preservedfor up to 72 hours by the preservative mixes; and 4) the absorbentmaterial did not irreversibly bind to the biomarkers. FIGS. 41, 42 and44 demonstrate consistency of biomarker preservation across twodifferent lots of SDJ.

ELISA Assay Compatibility

ELISA assays can be very sensitive and relatively complex immunoassaysthat can be negatively impacted by certain conditions. For example, anupstream component to an assay could inhibit or alter results. Theseexperiments were performed to characterize the impact of preservativecocktails on downstream biomarker assay methods and prompted a strategyof capturing materials for the immunoglobulin tests and the IL6 test inseparate ingestible devices.

As shown in FIG. 45, using 0.3% Cidal Mix 1, 1×HALT and 1% BSA resultedin successful IL6 detection in SDJ. As shown in FIGS. 46 and 47, bothIgA and IgM detection were inhibited by the presence of 0.3% CidalMix 1. Further investigation identified the sorbic acid and citric acidcomponents in Cidal Mix 1 were responsible for the IgA and IgM ELISAassay inhibition.

TPCK is a relatively stable and relatively irreversible inhibitor ofserine proteases and was included in preservation mixes to provideadditional protection against enzyme degradation. Studies were performedto determine the impact of TPCK's addition to the preservatives mixes.As shown in FIGS. 48-50, TPCK had no impact on the detection of IgA, IgMor IL-6, respectively.

Extraction of Protein Analytes in SDJ

Different extraction buffers and methods were tested to establish aprotocol which yielded effective recovery of analytes from SDJ.

IgA protein was detected by ELISA following extraction using both HyCultand Epitope extraction buffers, with Epitope exhibiting superiorresults. IL6 signal was observed following extraction using both Epitopeand Hycult extraction buffers. However, as shown in FIG. 51, IL6 wasdetected in heat treated SDJ. As shown in FIG. 52, IgA was detected inboth neat SDJ and heat treated SDJ. Extraction of IgM from SDJ waseffective with Epitope extraction buffer but not with Hycult extractionbuffer. As shown in FIG. 53, IgM was detectable in both neat and heattreated SDJ following extraction with Epitope buffer. As shown in FIG.54, IC was detectable in both neat and heat treated SDJ followingextraction with Epitope buffer.

Evaluation of Sponge Materials

Sponges made of different absorbent materials were tested for theirability to retain bacteria over time, and to determine their suitabilityfor use in an ingestible device. A sponge based on alginate,carboxymethyl cellulose, and collagene/cellulose was tested. Inparticular, the recovery of bacteria from sponges made from Promorgran™(Systagenix), Aquacel™ (Convatec), Nu-DERM™ (Systagenix) was tested overtime. Promorgran™ and Aquacel™ were rejected from consideration beingbecause Staphylococcus and Streptococcus bacteria could not be recoveredfrom these materials after they were seeded with bacteria. Nu-DERM™ wasalso rejected because gram positive strains could not be recovered after24 hours of incubation, and gram negative strains were significantlyreduced on this material over time. FIG. 55 shows the amounts ofStaphylococcus (F1) and Streptococcus (F6) on Nu-Derm™ over time.

Non-degradable synthetic matrix sponges were also tested to determine ifseeded bacteria could be recovered from them over time. Specifically,polyurethane (PU) and carbon sponges (C60 (60 ppi) and C100 (100 ppi))were seeded with E. coli (F1), Shigella (F6), or Staphylococcus aureus,and the recovery of bacteria was then tested over the course of 48hours. FIG. 56A-56C show that the polyurethane sponges allowed moreeffective recovery of all bacteria tested. Synthetic sponges made ofpolyurethane were also selected because they do not significantly changesize upon hydration, and preservatives can be stably added to thesponge.

The rate of saturation and final saturation weights for polyurethanesponges were then evaluated. Sponges were treated with Tween 80 andplaced in a thin layer of porcine duodenal fluid, and then time tosaturation and final weight were measured over time. FIG. 57 shows theabsorption of duodenal fluid by tween 80 treated polyurethane sponges.Complete saturation of the absorbent material was reached within 3minutes in all cases, and the weight of liquid absorbed is at least 30times the weight of the sponge.

Chemical Stabilization of Bacterial Cells for Flow Cytometry

Preservatives were tested on populations of bacteria to determine ifthey could stabilize bacterial cell counts over time. Bacteria cultureswere seeded with thimerosal, diazolidinyl urea, or imidazolidinyl urea,and cell counts were measured over the course of 72 hours using flowcytometry. FIG. 58 shows that each of the preservatives can inhibit orpreserve bacterial population growth for at least 72 hours. By contrast,control samples lacking preservative showed significant populationgrowth over time.

Quantification of Stabilized Bacterial Samples by PCR and Flow Cytometry

To test the effectiveness of preservatives for stabilizing bacterialpopulations, multiple bacterial strains were seeded with a testpreservative, and then bacteria were quantified over time using eitherPCR or FACS analysis. Test samples were prepared by inoculating strainsof bacteria in culture, combining the strain cultures, and then dilutingthe combined cultures in simulated duodenal fluid. An absorbent material(polyurethane sponge) was then treated with a preservative, and thebacterial sample was then loaded onto the absorbent material containingthe preservative, and incubated in a sealed tube at 37° C. for between24 hours and up to 8 days. The sample was then recovered and bacterialcells were quantified. Porcine duodenal fluid and canine duodenal fluidswere also tested using this assay. Porcine duodenal fluid and canineduodenal fluid were collected through laparotomy and biopsy of duodenalcontent.

Bactericidal preservatives were added to cultures containing bacterialcells at 4.5×10⁶ (high concentration) or at 8.6×10⁴ (low concentration),and then cell counts were quantified using PCR or plating over time, andcompared. The preservatives tested included TENT (Tris 50 mM, EDTA 50mM, NaCl 1%, and Tween 80 2.5%), with sorbic acid, thimerosal, or2-phenoxyethanol. FIGS. 59A and 59B show that the bactericidalpreservatives reduce the viability of bacteria in both the highconcentration and low concentration cultures.

Bactericidal preservatives also significantly reduced viability ofbacteria when measured by flow cytometry. Thimerosol, diazolidinyl urea,or imidazolinidyl urea were added to bacteria, and then cell counts wereassessed over 3 days using with flow cytometry or cell plating. FIG. 60shows that the bactericidal preservatives reduced the viability ofbacteria over time.

In both the PCR and flow cytometry assays, the cell counts of thebacteria in the presence of bactericidal preservatives corresponded tothe initial bacterial counts as assessed by plating (at time 0).

Other Embodiments

For illustrative purposes, the examples provided by above focusprimarily on a number of different exemplary embodiments of aningestible device. However, it is understood that variations in thegeneral shape and design of one or more embodiments of the ingestibledevices described herein (e.g., relation to the figures of devices) maybe made without significantly changing the functions and operations ofthe device. Furthermore, it should be noted that the features andlimitations described in any one embodiment may be applied to any otherembodiment herein, and the descriptions and examples relating to oneembodiment may be combined with any other embodiment in a suitablemanner. For example, any of the valves described in relation to FIG. 7may be used as the valves 214 and 216 described in relation to FIG. 2.As an alternate example, the absorptive material 310 and flexiblemembrane 314 described in relation to FIG. 3 may be incorporated intoany of the various sampling chambers described in various embodiments ofingestible devices 100, 200, 600, and 702-706 in order to automaticallyseal the sampling chamber. Moreover, the figures and examples providedin disclosure are intended to be only exemplary, and not limiting. Itshould also be noted that the systems and/or methods described above maybe applied to, or used in accordance with, other systems and/or methods,including systems and/or methods that may or may not be directly relatedto ingestible devices.

For illustrative purposes the disclosure focuses primarily on a numberof different example embodiments of an ingestible device, and exampleembodiments of methods for obtaining a sample when the ingestible devicewithin a GI tract. However, the possible ingestible devices that may beconstructed are not limited to these embodiments, and variations in theshape and design may be made without significantly changing thefunctions and operations of the device.

At least some of the elements of the various embodiments of theingestible device described herein that are implemented via software(e.g., software executed by control circuitry within PCB 120 (FIG. 2))may be written in a high-level procedural language such as objectoriented programming, a scripting language or both. Accordingly, theprogram code may be written in C, C⁺⁺ or any other suitable programminglanguage and may comprise modules or classes, as is known to thoseskilled in object oriented programming. Alternatively, or in addition,at least some of the elements of the embodiments of the ingestibledevice described herein that are implemented via software may be writtenin assembly language, machine language or firmware as needed. In eithercase, the language may be a compiled or an interpreted language.

At least some of the program code used to implement the ingestibledevice can be stored on a storage media or on a computer readable mediumthat is readable by a general or special purpose programmable computingdevice having a processor, an operating system and the associatedhardware and software that to implement the functionality of at leastone of the embodiments described herein. The program code, when read bythe computing device, configures the computing device to operate in anew, specific and predefined manner in order to perform at least one ofthe methods described herein.

Furthermore, at least some of the programs associated with the systems,devices, and methods of the example embodiments described herein arecapable of being distributed in a computer program product comprising acomputer readable medium that bears computer usable instructions for oneor more processors. The medium may be provided in various forms,including non-transitory forms such as, but not limited to, one or morediskettes, compact disks, tapes, chips, and magnetic and electronicstorage. In some embodiments, the medium may be transitory in naturesuch as, but not limited to, wire-line transmissions, satellitetransmissions, internet transmissions (e.g. downloads), media, digitaland analog signals, and the like. The computer useable instructions mayalso be in various formats, including compiled and non-compiled code.

The techniques described above can be implemented using software forexecution on a computer. For instance, the software forms procedures inone or more computer programs that execute on one or more programmed orprogrammable computer systems (which may be of various architecturessuch as distributed, client/server, or grid) each including at least oneprocessor, at least one data storage system (including volatile andnon-volatile memory and/or storage elements), at least one input deviceor port, and at least one output device or port.

The software may be provided on a storage medium, such as a CD-ROM,readable by a general or special purpose programmable computer ordelivered (encoded in a propagated signal) over a communication mediumof a network to the computer where it is executed. All of the functionsmay be performed on a special purpose computer, or using special-purposehardware, such as coprocessors. The software may be implemented in adistributed manner in which different parts of the computation specifiedby the software are performed by different computers. Each such computerprogram is preferably stored on or downloaded to a storage media ordevice (e.g., solid state memory or media, or magnetic or optical media)readable by a general or special purpose programmable computer, forconfiguring and operating the computer when the storage media or deviceis read by the computer system to perform the procedures describedherein. The inventive system may also be considered to be implemented asa computer-readable storage medium, configured with a computer program,where the storage medium so configured causes a computer system tooperate in a specific and predefined manner to perform the functionsdescribed herein.

1. An ingestible device, comprising: a housing defined by a first end, asecond end substantially opposite from the first end, and a wallextending longitudinally from the first end to the second end; a firstopening in the wall of the housing; a second opening in the first end ofthe housing, the second opening being oriented substantiallyperpendicular to the first opening; and a curved chamber connecting thefirst opening and the second opening, wherein at least a portion of thecurved chamber forms a sampling chamber within the ingestible device.2.-65. (canceled)
 66. An ingestible device having an opening between aninterior of the ingestible device and an exterior of the ingestibledevice, the ingestible device comprising: a chamber; and a multi-stagevalve system in the interior of the ingestible device, wherein: themulti-stage valve system has first, second and third states, the firststate of the multi-stage valve system is different from the second andthird states of the multi-stage valve system; the second state of themulti-stage valve system is different from the first and third states ofthe multi-stage valve system; when the multi-stage valve system is inits first state, the opening prevents fluid communication between theinterior of the ingestible device and the exterior of the ingestibledevice; when the multi-stage valve system is in its second state, theopening allows fluid communication between the interior of theingestible device and the exterior of the ingestible device; and whenthe multi-stage valve system is in its third state, the opening preventsfluid communication between the interior of the ingestible device andthe exterior of the ingestible device.
 67. The ingestible device ofclaim 66, wherein: the multi-stage valve system comprises an actuatorsystem having first, second and third states; when the actuator systemis in its first state, the multi-stage valve system is in its firststate; when the actuator system is in its second state, the multi-stagevalve system is in its second state; and when the actuator system is inits third state, the multi-stage valve system is in its third state. 68.The ingestible device of claim 67, wherein the actuator system comprisesfirst and second members.
 69. The ingestible device of claim 68,wherein: when the multi-stage valve system is in its first stage, thefirst member holds the multi-stage system in its first state; and whenthe multi-stage system is in its second stage, the second member holdsthe valve multi-stage system in its second state.
 70. The ingestibledevice of claim 68, wherein the first member comprises a first chambercomprising wax, and the second member comprises a second chambercomprising wax.
 71. The ingestible device of claim 69, wherein: when theactuator system is in its first state, the wax in the first chamber issolid; and when the actuator system is configured so that, when theactuator is changing from its first state to its second stage in itssecond state, at least a portion of the wax in the first chamber isliquid.
 72. The ingestible device of claim 70, further comprising adevice configured to heat the wax in the first chamber.
 73. Theingestible device of claim 70, wherein: when the actuator system is inits second state, the wax in the second chamber is solid; and when theactuator system is configured so that, when the actuator is changingfrom its second state to its second stage in its third state, at least aportion of the wax in the second chamber is liquid.
 74. The ingestibledevice of claim 70, further comprising a device configured to heat thewax in the second chamber.
 75. The ingestible device of claim 67,wherein the multi-stage valve system further comprises a triggermechanically coupled with the actuator system.
 76. The ingestible deviceof claim 75, wherein: the trigger has first, second and third states;when the actuator system is in its first state, the trigger is in itsfirst state; when the actuator system is in its second state, thetrigger is in its second state; and when the actuator system is in itsthird state, the trigger is in its third state.
 77. The ingestibledevice of claim 67, wherein the valve system further comprises a gatemechanically coupled to the actuator system.
 78. The ingestible deviceof claim 77, wherein: the gate has first, second and third states; whenthe actuator system is in its first state, the gate is in its firststate; when the actuator system is in its second state, the gate is inits second state; and when the actuator system is in its third state,the gate is in its third state.
 79. The ingestible device of claim 77,wherein: the gate has an opening; when the gate is in its first state,the opening of gate and the opening of the ingestible device are notaligned; and when the gate is in its second state, the opening of gateand the opening of the ingestible device are aligned; when the gate isin its third state, the opening of gate and the opening of theingestible device are not aligned.
 80. The ingestible device of claim67, wherein the multi-stage valve system further comprises a biasingsystem mechanically coupled to actuator system.
 81. The ingestibledevice of claim 80, wherein the biasing system comprises first andsecond biasing members.
 82. The ingestible device of claim 81, whereinfirst member comprises a first spring, and the second member comprises asecond spring.
 83. The ingestible device of claim 66, further comprisinga sampling system configured so that, when the valve system is in itssecond stage, the exterior of the ingestible device is in fluidcommunication with the sampling system.
 84. The ingestible device ofclaim 83, wherein the sampling system comprises a plurality of absorbentmembers.
 85. The ingestible device of claim 83, wherein the samplingsystem comprises a biomarker preservative.
 86. The ingestible device ofclaim 66, further comprising an analytical system configured to analyzea sample in the interior of the ingestible device.
 87. The ingestibledevice of claim 66, further comprising a microprocessor configured tocontrol at least one system of the ingestible device.
 88. An ingestibledevice having an opening between an interior of the ingestible deviceand an exterior of the ingestible device, the ingestible devicecomprising: a chamber; and a multi-stage valve system in the interior ofthe ingestible device, wherein: the multi-stage valve system comprises:an actuator system comprising a first member; a trigger comprising afirst peg and a first lip; a gate comprising a protrusion, and a gateleg having an opening; and a biasing system comprising first and secondbiasing members; when the multi-stage valve system is in a first stage:the first biasing member applies a force to the trigger so that thefirst peg contacts the first member; the first member opposes the forceapplied to the trigger by the first biasing member; the second biasingmember applies a force to the gate so that the protrusion contacts thefirst lip; the first lip opposes the force applied to the gate by thesecond biasing member; and the opening in the gate leg is not alignedwith the opening in the ingestible device. 89.-100. (canceled)
 101. Aningestible device having an opening between an interior of theingestible device and an exterior of the ingestible device, theingestible device comprising: a chamber; and a sampling system in theinterior of the ingestible device, wherein: the sampling systemcomprises: a first absorbent member; and a second absorbent memberdifferent from the first absorbent member; and the sampling system isconfigured so that fluid that flows from the exterior of the ingestibledevice to the interior of the ingestible device enters the firstabsorbent member; and the sampling system is configured to allow fluidto flow from the first absorbent member to the second absorbent member.102.-115. (canceled)
 116. An ingestible device having an opening betweenan interior of the ingestible device and an exterior of the ingestibledevice, the ingestible device comprising: a chamber; and a samplingsystem in the interior of the ingestible device configured to absorb afluid that enters the interior of the ingestible device via the opening,the sampling system comprising an absorbent member and at least onepreservative at least partially absorbed in the absorbent member.117.-150. (canceled)
 151. A method, comprising: collecting a sample intoa sampling system of an ingestible device, wherein the sampling systemcomprises an absorbent member and at least one preservative at leastpartially absorbed in the absorbent member. 152.-186. (canceled)